Adipocyte-specific protein homologs

ABSTRACT

The present invention relates to polynucleotide and polypeptide molecules for zsig39, a novel member of the family of proteins bearing a collagen-like domain and a globular domain. The polypeptides, and polynucleotides encoding them, are involved in dimerization or oligomerization and may be used in the study thereof. The present invention also includes antibodies to the zsig39 polypeptides.

REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser.No. 09/140,804, filed Aug. 26, 1998, which is related to ProvisionalApplication 60/056,983, filed on Aug. 26, 1997. Under 35 U.S.C.§119(e)(1), this application claims benefit of said Applications.

BACKGROUND OF THE INVENTION

[0002] Energy balance (involving energy metabolism, nutritional state,lipid storage and the like) is an important criteria for health. Thisenergy homeostasis involves food intake and metabolism of carbohydratesand lipids to generate energy necessary for voluntary and involuntaryfunctions. Metabolism of proteins can lead to energy generation, butpreferably leads to muscle formation or repair. Among otherconsequences, a lack of energy homeostasis lead to over or underformation of adipose tissue.

[0003] Formation and storage of fat is insulin-modulated. For example,insulin stimulates the transport of glucose into cells, where it ismetabolized into α-glycerophosphate which is used in the esterificationof fatty acids to permit storage thereof as triglycerides. In addition,adipocytes (fat cells) express a specific transport protein thatenhances the transfer of free fatty acids into adipocytes.

[0004] Adipocytes also secrete several proteins believed to modulatehomeostatic control of glucose and lipid metabolism. These additionaladipocyte-secreted proteins include adipsin, complement factors C3 andB, tumor necrosis factor α, the ob gene product and Acrp30. Evidencealso exists suggesting the existence of an insulin-regulated secretorypathway in adipocytes. Scherer et al., J. Biol. Chem. 270(45): 26746-9,1995. Over or under secretion of these moieties, impacted in part byover or under formation of adipose tissue, can lead to pathologicalconditions associated directly or indirectly with obesity or anorexia.

[0005] Acrp30is a 247 amino acid polypeptide that is expressedexclusively by adipocytes. The Acrp30 polypeptide is composed of aamino-terminal signal sequence, a 27 amino acid stretch of no knownhomology, 22 perfect Gly-Xaa-Pro or imperfect Gly-Xaa-Xaa collagenrepeats and a carboxy terminal globular domain. See, Scherer et al. asdescribed above and International Patent Application No. WO96/39429.Acrp30, an abundant human serum protein regulated by insulin, sharesstructural similarity, particularly in the carboxy-terminal globulardomain, to complement factor Clq and to a summer serum protein ofhibernating Siberian chipmunks (Hib27). Expression of Acrp30 is inducedover 100-fold during adipocyte differentiation. Acrp30 is suggested foruse in modulating energy balance and in identifying adipocytes in testsamples.

[0006] Another secreted protein that appears to be exclusively producedin adipocytes is apM1, described, for example, in Maeda et al., Biochem.Biophys. Res. Comm. 221: 286-9, 1996. A 4517 bp clone had a 244 aminoacid open reading frame and a long 3′ untranslated region. The proteinincluded a signal sequence, an amino-terminal non-collagenous sequence,22 collagen repeats (Gly-XAA-Pro or Gly-Xaa-Xaa), and a carboxy-terminalregion with homology to collagen X, collagen VIII and complement proteinClq.

[0007] Complement factor Clq consists of six copies of three relatedpolypeptides (A, B and C chains), with each polypeptide being about 225amino acids long with a near amino-terminal collagen domain and acarboxy-terminal globular region. Six triple helical regions are formedby the collagen domains of the six A, six B and six C chains, forming acentral region and six stalks. A globular head portion is formed byassociation of the globular carboxy terminal domain of an A, a B and a Cchain. Clq is therefore composed of six globular heads linked via sixcollagen-like stalks to a central fibril region. Sellar et al., Biochem.J. 274: 481-90, 1991. This configuration is often referred to as abouquet of flowers. Acrp30 has a similar bouquet structure formed from asingle type of polypeptide chain.

[0008] Molecules capable of modulating energy homeostasis are sought forthe study of this phenomena and for the prevention or treatment ofimbalances. Also, molecules capable of modulating adipocyte secretorypathways are also sought as indirect energy homeostasis modulators andas research reagents.

[0009] The present invention provides such polypeptides for these andother uses that should be apparent to those skilled in the art from theteachings herein.

SUMMARY OF THE INVENTION

[0010] Within one aspect of the invention is provided an isolatedpolypeptide comprising a sequence of amino acid residues that is atleast 80% identical to SEQ ID NO:2, wherein the sequence comprises: betastrands corresponding to amino acid residues 105-109, 128-130, 136-139,143-146, 164-171, 176-182, 187-200, 204-210 and 226-231 of SEQ ID NO:2,wherein the beta strands are separated by at least two amino acidresidues; and a receptor binding domain comprising amino acid residues111-135 and 170-174 of SEQ ID NO:2. Within one embodiment thepolypeptide is at least 90% identical to SEQ ID NO:2. Within anotherembodiment the polypeptide comprises a collagen-like domain having atleast 22 collagen repeats. Within another embodiment the polypeptidecomprises residues, 19-243 of SEQ ID NO:2. Within yet another embodimentthe polypeptide is covalently linked amino terminally or carboxyterminally to a moiety selected from the group consisting of affinitytags, toxins, radionucleotides, enzymes and fluorophores.

[0011] Within another aspect is provided an isolated polypeptideselected from the group consisting of: a) a polypeptide having asequence of amino acid residues from amino acid residue 30 to amino acidresidue 95 of SEQ ID NO:2; b) a polypeptide having a sequence of aminoacid residues from amino acid residue 30 to amino acid residue 96 of SEQID NO:2; and c) a polypeptide having a sequence of amino acid residuesfrom amino acid residue 30 to 97 of SEQ ID NO:2; d) a polypeptide havinga sequence of amino acid residues from amino acid residue 30 to aminoacid residue 98 of SEQ ID NO: 2; e) a polypeptide having a sequence ofamino acid residues from amino acid residue 98 to amino acid residue 243of SEQ ID NO:2; f) a polypeptide having a sequence of amino acidresidues from amino acid residue 99 to amino acid residue 243 of SEQ IDNO:2; g) a polypeptide having a sequence of amino acid residues fromamino acid residue 30 to amino acid residue 243 of SEQ ID NO:2; and h) apolypeptide having a sequence of amino acid residues that is 90%identical in amino acid sequence to a), b), c), d), e), f), g) or h).

[0012] Within another aspect is provided a fusion protein consistingessentially of a first portion and a second portion joined by a peptidebond, the first portion comprising a polypeptide selected from the groupconsisting of: a) a polypeptide comprising a sequence of amino acidresidues that is at least 800 identical to SEQ ID NO:2, wherein thesequence comprises: beta strands corresponding to amino acid residues105-109, 128-130, 136-139, 143-146, 164-171, 176-182, 187-200, 204-210and 226-231 of SEQ ID NO:2, wherein the beta strands are separated by atleast two amino acid residues; and a receptor binding domain comprisingamino acid residues 111-135 and 170-174 of SEQ ID NO:2; b) a polypeptidecomprising a sequence of amino acid residues as shown in SEQ ID NO:2from amino acid residue 16 to amino acid residue 243; c) a polypeptidecomprising a sequence of amino acid residues as shown in SEQ ID NO:2from amino-acid residue 1 to amino acid residue 243; d) a portion of thezsig39 polypeptide as shown in SEQ ID NO:2 containing the collagen-likedomain or a portion of the collagen-like domain capable of dimerizationor oligomerization; e) a portion of the zsig39 polypeptide as shown inSEQ ID NO:2, containing the globular-like domain or the receptor bindingportion of the globular-like domain; or f) a portion of the zsig39polypeptide as shown in SEQ ID NO:2, including the collagen-like domainand the globular domain; and the second portion comprising anotherpolypeptide. Within one embodiment the first portion is selected fromthe group consisting of: a) a polypeptide having the sequence of aminoacid residue 30 to amino acid residue 95 of SEQ ID NO:2; b) apolypeptide having the sequence of amino acid residue 30 to amino acidresidue 96 of SEQ ID NO:2; c) a polypeptide having the sequence of aminoacid residue 30 to amino acid residue 97 of SEQ ID NO:2; d) apolypeptide having the sequence of amino acid residue 30 to amino acidresidue 98 of SEQ ID NO:2; e) a polypeptide having the sequence of aminoacid residue 30 to amino acid residue 243 of SEQ ID NO:2; f) apolypeptide having the sequence of amino acid residue 98 to amino acidresidue 243 of SEQ ID NO:2; and g) a polypeptide having the sequence ofamino acid residue 99 to amino acid residue 243 of SEQ ID NO:2.

[0013] Within another aspect is provided a fusion protein comprising asecretory signal sequence having the amino acid sequence of amino acidresidues 1-15 or 1-18 of SEQ ID NO:2, wherein the secretory signalsequence is operably linked to an additional polypeptide.

[0014] Within yet another aspect is pharmaceutical compositioncomprising a polypeptide as described above, in combination with apharmaceutically acceptable vehicle.

[0015] Also provided is an antibody that specifically binds to anepitope of a polypeptide as described above.

[0016] Further provided is an isolated polynucleotide encoding apolypeptide comprising a sequence of amino acid residues that is atleast 809s identical to SEQ ID NO:2, wherein the sequence comprises:beta strands corresponding to amino acid residues 105-109, 128-130,136-139, 143-146, 164-171, 176-182, 187-200, 204-210 and 226-231 of SEQID NO:2, wherein the beta strands are separated by at least two aminoacid residues; and a receptor binding domain comprising amino acidresidues 111-135 and 170-174 of SEQ ID NO:2. Within one embodiment thepolypeptide is at least 900- identical to SEQ ID NO:2. Within anotherembodiment the polypeptide comprises a collagen-like domain having atleast 22 collagen repeats. Within another embodiment the polynucleotideis DNA.

[0017] Within yet another aspect is provided an isolated polynucleotideselected from the group consisting of: a) a sequence of nucleotides fromnucleotide 243 to nucleotide 962 of SEQ ID NO:1; b) a sequence ofnucleotides from nucleotide 252 to nucleotide 962 of SEQ ID NO:1; c) asequence of nucleotides from nucleotide 285 to nucleotide 482 of SEQ IDNO:1; d) a sequence of nucleotides from nucleotide 285 to nucleotide 485of SEQ ID NO:1; e) a sequence of nucleotides from nucleotide 285 tonucleotide 488 of SEQ ID NO:1; f) a sequence of nucleotides fromnucleotide 285 to nucleotide 491 of SEQ ID NO:1; g) a sequence ofnucleotides from nucleotide 285 to nucleotide 926 of SEQ ID NO:1; h) asequence of nucleotides from nucleotide 491 to nucleotide 926 of SEQ IDNO:1; i) a polynucleotide encoding a polypeptide having a sequence ofnucleotides that is at least 80% identical in nucleotide sequence to a),b), c), d), e), f), g) and h); j) nucleotide sequences complementary toa) , b), c), d), e), f), g), h) or i); and k) degenerate nucleotidesequences of a), b), c), d), e), f), g), h), i) or j).

[0018] Within another aspect is provided an isolated polynucleotideencoding a fusion protein consisting-essentially of a first portion anda second portion joined by a peptide bond, the first portion is selectedfrom the group consisting of: a) a polypeptide comprising a sequence ofamino acid residues that is at least 80% identical to SEQ ID NO: 2,wherein the sequence comprises: beta strands corresponding to amino acidresidues 105-109, 128-130, 136-139, 143-146, 164-171, 176-182, 187-200,204-210 and 226-231 of SEQ ID NO:2, wherein the beta strands areseparated by at least two amino acid residues; and a receptor bindingdomain comprising amino acid residues 111-135 and 170-174 of SEQ IDNO:2; b) a polypeptide comprising a sequence of amino acid residues asshown in SEQ ID NO:2 from amino acid residue 16 to amino acid residue243; c) a polypeptide comprising a sequence of amino acid residues asshown in SEQ ID NO:2 from amino acid residue 1 to amino acid residue243; d) a portion of the zsig39 polypeptide as shown in SEQ ID NO:2containing the collagen-like domain or a portion of the collagen-likedomain capable of dimerization or oligomerization; e) a portion of thezsig39 polypeptide as shown in SEQ ID NO:2, containing the globular-likedomain or an active portion of the globular-like domain; or f) a portionof the zsig39 polypeptide as shown in SEQ ID NO:2, including thecollagen-like domain and the globular domain; and the second portioncomprising another polypeptide.

[0019] Within another aspect is provided an isolated polynucleotideencoding a fusion protein comprising a secretory signal sequence havingthe amino acid sequence of amino acid residues 1-15 or 1-18 of SEQ IDNO:2, wherein the secretory signal sequence is operably linked to anadditional polypeptide.

[0020] Within yet another aspect is an isolated polynucleotidecomprising the sequence of nucleotide 1 to nucleotide 729 of SEQ IDNO:10.

[0021] Also provided is an expression vector comprising the followingoperably linked elements: a transcription-promoter; a DNA segmentencoding a polypeptide as described above; and a transcriptionterminator. Within one embodiment the DNA segment encodes a polypeptidethat is at least 90% identical to SEQ ID NO:2. Within another embodimentthe DNA segment encodes a polypeptide further comprising a collagen-likedomain having at least 22 collagen repeats. Within yet anotherembodiment the DNA segment encodes a polypeptide covalently linked aminoterminally or carboxy terminally to an affinity tag. Within stillanother embodiment the DNA segment further encodes a secretory signalsequence operably linked to the polypeptide. Within yet anotherembodiment the secretory signal sequence comprises residues 1-15 or 1-18of SEQ ID NO:2.

[0022] Also provided is a cultured cell into which has been introducedan expression vector comprising the following operably linked elements:a transcription promoter; a DNA segment encoding a polypeptide asdescribed above; and a transcription terminator; wherein the cellexpresses the polypeptide encoded by the DNA segment.

[0023] Within another aspect is provided a method of producing apolypeptide comprising: culturing a cell into which has been introducedan expression vector comprising the following operably linked elements:a transcription promoter; a DNA segment encoding a polypeptide asdescribed above; and a transcription terminator; whereby the cellexpresses the polypeptide encoded by the DNA segment; and recovering theexpressed polypeptide.

[0024] Within another aspect is an oligonucleotide probe or primercomprising at least 14 contiguous nucleotides of a polynucleotide of SEQID NO:10 or a sequence complementary to SEQ ID NO:10.

[0025] Within yet another aspect is a method for modulating free fattyacid metabolism by administering a pharmaceutically effective dose of apolypeptide as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1 illustrates a multiple alignment of and zsig39 polypeptideof the present invention and HUMUPST2_(—)1 (SEQ ID NO:3) (Maeda et al.,Biochem. Biophys. Res. Comm. 221(2): 286-9, 1996); C1QA_HUMAN (SEQ IDNO:4) (Sellar et al., Biochem. J. 274: 481-90, 1991, Reid, Biochem. J.179: 367-71, 1979, and Reid et al., Biochem. J. 203: 559-69, 1982);HP25_TAMAS (SEQ ID NO:5) (Takamatsu et al., Mol. Cell. Biol. 13:1516-21, 1993 and Kondo & Kondo, J. Biol. Chem. 267: 473-8, 1992);HP27_TAMAS (SEQ ID NO:6) (Takamatsu et al. and Kondo & Kondo referencedabove); and CERL_RAT (SEQ ID NO:7)(Wada & Ohtani, Brain Res. Mol. BrainRes. 9: 71-7, 1991).

[0027]FIG. 2 is a matrix showing percent amino acid identity in acomparison of the six proteins shown in the multiple alignment FIG. 1.

DETAILED DESCRIPTION OF THE INVENTION

[0028] Prior to setting forth the invention in detail, it may be helpfulto the understanding thereof to define the following terms.

[0029] The term “affinity tag” is used herein to denote a polypeptidesegment that can be attached to a second polypeptide to provide forpurification or detection of the second polypeptide or provide sites forattachment of the second polypeptide to a substrate. In principal, anypeptide or protein for which an antibody or other specific binding agentis available can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), substance P, Flag™ peptide (Hoppet al., Biotechnology 6:1204-1210, 1988; available from Eastman KodakCo., New Haven, Conn.), Glu-Glu affinity tag (Grussenmeyer et al., Proc.Natl. Acad. Sci. USA 82:7925-4, 1995), streptavidin binding peptide, orother antigenic epitope or binding domain. See, in general Ford et al.,Protein Expression and Purification 2: 95-107, 1991. DNAs encodingaffinity tags are available from commercial suppliers (e.g., PharmaciaBiotech, Piscataway, N.J.).

[0030] The term “allelic variant” denotes any of two or more alternativeforms of a gene occupying the same chromosomal locus. Allelic variationarises naturally through mutation, and may result in phenotypicpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequence. The term allelic variant is also used herein todenote a protein encoded by an allelic variant of a gene.

[0031] The terms “amino-terminal” and “carboxyl-terminal” are usedherein to denote positions within polypeptides and proteins. Where thecontext allows, these terms are used with reference to a particularsequence or portion of a polypeptide or protein to denote proximity orrelative position. For example, a certain sequence positionedcarboxyl-terminal to a reference sequence within a protein is locatedproximal to the carboxyl terminus of the reference sequence, but is notnecessarily at the carboxyl terminus of the complete protein.

[0032] The term “collagen or collagen-like domain” refers to a series ofrepeating triplet amino acid sequences,, “repeats” or “collagenrepeats”, Gly-Xaa-Pro or Gly-Xaa-Xaa, where Xaa is any amino acidresidue. Such domains may contain as many as 22 collagen repeats ormore. Fragments or proteins containing such collagen-like domains mayform homomeric constructs (dimers or oligomers of the same fragment orprotein). Moreover, such fragments or proteins containing suchcollagen-like domains may form heteromeric constructs (dimers oroligomers of different fragments or proteins).

[0033] The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

[0034] The term “complements of polynucleotide molecules” denotespolynucleotide molecules having a complementary base sequence andreverse orientation as compared to a reference sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

[0035] The term “contig” denotes a polynucleotide that has a contiguousstretch of identical or complementary sequence to anotherpolynucleotide. Contiguous sequences are said to “overlap” a givenstretch of polynucleotide sequence either in their entirety or along apartial stretch of the polynucleotide. For example, representativecontigs to the polynucleotide sequence 5′-ATGGCTTAGCTT-3′ are5′-TAGCTTgagtct-3′ and 3′-gtcgacTACCGA-5′.

[0036] The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

[0037] The term “expression vector” denotes a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments may include promoter andterminator sequences, and may optionally include one or more origins ofreplication, one or more selectable markers, an enhancer, apolyadenylation signal, and the like. Expression vectors are generallyderived from plasmid or viral DNA, or may contain elements of both.

[0038] The term “isolated”, when applied to a polynucleotide, denotesthat the polynucleotide has been removed from its natural genetic milieuand is thus free of other extraneous or unwanted coding sequences, andis in a form suitable for use within genetically engineered proteinproduction systems. Such isolated molecules are those that are separatedfrom their natural environment and include cDNA and genomic clones.Isolated DNA molecules of the present invention are free of other geneswith which they are ordinarily associated, but may include naturallyoccurring 5′ and 3′ untranslated regions such as promoters andterminators. The identification of associated regions will be evident toone of ordinary skill in the art (see for example, Dynan and Tijan,Nature 316:774-78, 1985).

[0039] An “isolated” polypeptide or protein is a polypeptide or proteinthat is found in a condition other than its native environment, such asapart from blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

[0040] The term “operably linked”, when referring to DNA segments,denotes that the segments are arranged so that they function in concertfor their intended purposes, e.g. transcription initiates in thepromoter and proceeds through the coding segment to the terminator.

[0041] The term “ortholog” denotes a polypeptide or protein obtainedfrom one species that is the functional counterpart of a polypeptide orprotein from a different species. Sequence differences among orthologsare the result of speciation.

[0042] “Paralogs” are distinct but structurally related proteins made byan organism. Paralogs are believed to arise through gene duplication.For example, α-globin, β-globin, and myoglobin are paralogs of eachother.

[0043] The term “polynucleotide” denotes a single- or double-strandedpolymer of deoxyribonucleotide or ribonucleotide bases read from the 5′to the 3′ end. Polynucleotides include RNA and DNA, and may be isolatedfrom natural sources, synthesized in vitro, or prepared from acombination of natural and synthetic molecules. Sizes of polynucleotidesare expressed as base pairs (abbreviated “bp”), nucleotides (“nt”), orkilobases (“kb”). Where the context allows, the latter two terms maydescribe polynucleotides that are single-stranded or double-stranded.When the term is applied to double-stranded molecules it is used todenote overall length and will be understood to be equivalent to theterm “base pairs”. It will be recognized by those skilled in the artthat the two strands of a double-stranded polynucleotide may differslightly in length and that the ends thereof may be staggered as aresult of enzymatic cleavage; thus all nucleotides within adouble-stranded polynucleotide molecule may not be paired. Such unpairedends will in general not exceed 20 nt in length.

[0044] A “polypeptide” is a polymer of amino acid residues joined bypeptide bonds, whether produced naturally or synthetically. Polypeptidesof less than about 10 amino acid residues are commonly referred to as“peptides”.

[0045] “Probes and/or primers” as used herein can be RNA or DNA. DNA canbe either cDNA or genomic DNA. Polynucleotide probes and primers aresingle or double-stranded DNA or RNA, generally syntheticoligonucleotides, but may be generated from cloned cDNA or genomicsequences or its complements. Analytical probes will generally be atleast 20 nucleotides in length, although somewhat shorter probes (14-17nucleotides) can be used. PCR primers are at least 5 nucleotides inlength, preferably 15 or more nt, more preferably 20-30 nt. Shortpolynucleotides can be used when a small region of the gene is targetedfor analysis. For gross analysis of genes, a polynucleotide probe maycomprise an entire exon or more. Probes can be labeled to provide adetectable signal, such as with an enzyme, biotin, a radionuclide,fluorophore, chemiluminescer, paramagnetic particle and the like, whichare commercially available from many sources, such as Molecular Probes,Inc., Eugene, Oreg., and Amersham Corp., Arlington Heights, Ill., usingtechniques that are well known in the art.

[0046] The term “promoter” denotes a portion of a gene containing DNAsequences that provide for the binding of RNA polymerase and initiationof transcription. Promoter sequences are commonly, but not always, foundin the 5′ non-coding regions of genes.

[0047] A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is. produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

[0048] The term “receptor” denotes a cell-associated protein that bindsto a bioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-domain structure comprising an extracellular ligand-binding domainand an intracellular effector domain that is typically involved insignal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. Most nuclear receptors also exhibit amulti-domain structure, including an amino-terminal, transactivatingdomain, a DNA binding domain and a ligand binding domain. In general,receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g.,thyroid stimulating hormone receptor, beta-adrenergic receptor) ormultimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor,GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6receptor).

[0049] The term “secretory signal sequence” denotes a DNA sequence thatencodes a polypeptide (a “secretory peptide”) that, as a component of alarger polypeptide, directs the larger polypeptide through a secretorypathway of a cell in which it is synthesized. The larger peptide iscommonly cleaved to remove the secretory peptide during transit throughthe secretory pathway.

[0050] A “soluble receptor” is a receptor polypeptide that is not boundto a cell membrane. Soluble receptors are most commonly ligand-bindingreceptor polypeptides that lack transmembrane and cytoplasmic domains.Soluble receptors can comprise additional amino acid residues, such asaffinity tags that provide for purification of the polypeptide orprovide sites for attachment of the polypeptide, to a substrate, orimmunoglobulin constant region sequences. Many cell-surface receptorshave naturally occurring, soluble counterparts that are produced byproteolysis or translated from alternatively spliced mRNAs. Receptorpolypeptides are said to be substantially free of transmembrane andintracellular polypeptide segments when they lack sufficient portions ofthese segments to provide membrane anchoring or signal transduction,respectively.

[0051] The term “splice variant” is used herein to denote alternativeforms of RNA transcribed from a gene. Splice variation arises naturallythrough use of alternative splicing sites within a transcribed RNAmolecule, or less commonly between separately transcribed RNA molecules,and may result in several mRNAs transcribed from the same gene. Splicevariants may encode polypeptides having altered amino acid sequence. Theterm splice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

[0052] Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

[0053] All references cited herein are incorporated by reference intheir entirety.

[0054] The present invention is based in part upon the discovery of anovel DNA sequence that encodes a polypeptide having homology to anadipocyte complement related protein (Acrp30) . See, for example,Scherer et al., J. Biol. Chem. 270(45): 26746-9, 1995. The polypeptideAcrp30 is shown in SEQ ID NO:8. Acrp30appears to be highly related tohuman apM1 (HUMUPST2_(—)1 in FIGS. 1 and 2, SEQ ID NO:3), with the mostsignificant differences observed in the secretory sequence.

[0055] The novel DNA sequence encodes a polypeptide having anamino-terminal signal sequence, an adjacent N-terminal region ofnon-homology, a truncated collagen domain composed of Gly-Xaa-Xaa orGly-Xaa-Pro repeats and a carboxy-terminal globular portion. The novelpolynucleotide sequence also contains a long 3′ untranslated region. Thegeneral polypeptide structure set forth above is shared by Acrp30 (SEQID NO:8) and HUMUPST2_(—)1 (SEQ ID NO:3). Also, the HUMUPST2_(—)1 DNAsequence (SEQ ID NO:9) is characterized by a long 3′ untranslatedregion. Moreover, Acrp30 and all of the sequences aligned in FIG. 1,with the exception of CERL_RAT (SEQ ID NO:7), share a conserved cysteineresidue at position 144 of the zsig39 polypeptide as shown in FIG. 1 andSEQ ID NO: 2. Other regions of homology, found in the carboxy-terminalglobular portion in the aligned proteins, are identified herein asuseful primers for searching for other family members. Acrp30, forexample, would be identified in a search using the primers. Also, thezsig39 polypeptides of the present invention include a putative cellattachment site, the RGD motif at amino acid residues 77-79 of SEQ IDNO: 2. See, for example, Ruoslahti and Pierschbacher, Cell 44: 517-8,1986 and d'Souza et al., Trends Biochem. Sci. 16: 246-50, 1991, fordiscussions of the RGD peptide motif and its role in adhesion.

[0056] Analysis of the tissue distribution of the mRNA corresponding tothis novel DNA was conducted as described in Example 2 herein. Onetranscript size was observed at approximately 1.2 kb. Signal intensitywas highest for small intestine and heart, with relatively less intensesignals in pancreas, skeletal muscle, kidney and thyroid, and with lowerintensity signals in placenta, lung, liver, spleen, prostate, ovary,colon, stomach, spinal cord, lymph node, trachea, adrenal gland and bonemarrow. The polypeptide has been designated zsig39 polypeptide. A Dotblot indicated expression of zsig39 polypeptide in the subthalamicnucleus, hippocampus, medulla oblongata and thalamus. A human gut blotshowed expression in the human colorectal adenocarcinoma cell lineSW480, small intestine tissue, stomach tissue, normal human colon cellline, FHC; and normal fetal small intestine cell line FHs74 Int.

[0057] The novel zsig39 polypeptides of the present invention wereinitially identified by querying an EST database for secretory signalsequences, characterized by an upstream methionine start site, ahydrophobic region of approximately 13 amino acids and a cleavage site,in an effort to select for secreted proteins. Polypeptides correspondingto ESTs meeting those search criteria were compared to known sequencesto identify secreted proteins having homology to known ligands. A singleEST sequence was discovered and predicted to be a secreted protein. Thenovel polypeptide encoded by the full length cDNA enable theidentification of a homolog relationship with adipocyte complementrelated protein Acrp30 (SEQ ID NO:8) and adipocyte secreted protein apM1(HUMUPST2_(—)1 in FIGS. 1 and 2, SEQ ID NO:3). Somewhat more distanthomology was also identified to complement component Clq A chain, twofactors observed in the active state of hibernating Siberian woodchucks(HP25_TAMAS (SEQ ID NO:5) and HP27_TAMAS (SEQ ID NO:6)) and a rat brainprotein (CERL_RAT, SEQ ID NO:7), as shown in FIGS. 1 and 2.

[0058] The full sequence of the zsig39 polypeptide was obtained from asingle clone believed to contain it, wherein the clone was obtained froma lung tissue library. Other libraries that might also be searched forsuch clones include heart, small intestine, pancreas, skeletal muscle,kidney, thyroid, subthalamic nucleus, hippocampus, medulla oblongata,thalamus and the like.

[0059] The nucleotide sequence of the N-terminal EST is described in SEQID NO:1, and its deduced amino acid sequence is described in SEQ IDNO:2. As described generally above, the zsig39 polypeptide includes asignal sequence, ranging from amino acid 1 (Met) to amino acid residue15 (Gly). An alternative signal sequence ranges from amino acid 1 (Met)to amino acid 18 (Pro). The mature polypeptide therefore ranges fromamino acid 16 (Ser) or 19 (Leu) to amino acid 243 (Ala). Within themature polypeptide, an N-terminal region of limited homology is found,ranging between amino acid residue 20 (Asp) and 29 (Pro), wherein thecysteine at position 28 may provide similar structure/function as thecysteine found at position 36 in HUMUPST2_(—)1 and in the N-terminalregion of HP25_TAMAS and HP27_TAMAS. In addition, a collagen domain isfound between amino acid 30 (Gly) and 95 (Ala), 96 (Gly), 97 (Glu) or 98(Cys). In the collagen domain, 9 perfect Gly-Xaa-Pro and 13 or 14imperfect Gly-Xaa-Xaa repeats are observed. Acrp30 contains 22 perfector imperfect repeats.

[0060] The zsig39 polypeptide also includes a carboxy-terminal globulardomain, ranging from about amino acid 98 the zsig39 polypeptides of thepresent invention include a putative cell attachment site, the RGD motifat amino acid residues 77-79 of SEQ ID NO: 2. See, for example,Ruoslahti and Pierschbacher, Cell 44: 517-8, 1986 and d'Souza et al.,Trends Biochem. Sci. 16: 246-50, 1991, for discussions of the RGDpeptide motif and its role in adhesion.

[0061] Analysis of the tissue distribution of the mRNA corresponding tothis novel DNA was conducted as described in Example 2 herein. Onetranscript size was observed at approximately 1.2 kb. Signal intensitywas highest for small intestine and heart, with relatively less intensesignals in pancreas, skeletal muscle, kidney and thyroid, and with lowerintensity signals in placenta, lung, liver, spleen, prostate, ovary,colon, stomach, spinal cord, lymph node, trachea, adrenal gland and bonemarrow. The polypeptide has been designated zsig39 polypeptide. A Dotblot indicated expression of zsig39 polypeptide in the subthalamicnucleus, hippocampus, medulla oblongata and thalamus. A human gut blotshowed expression in the human colorectal adenocarcinoma cell lineSW480, small intestine tissue, stomach tissue, normal human colon cellline, FHC; and normal fetal small intestine cell line FHs74 Int.

[0062] The novel zsig39 polypeptides of the present invention wereinitially identified by querying an EST database for secretory signalsequences, characterized by an upstream methionine start site, ahydrophobic region of approximately 13 amino acids and a cleavage site,in an effort to select for secreted proteins. Polypeptides correspondingto ESTs meeting those search criteria were compared to known sequencesto identify secreted proteins having homology to known ligands. A singleEST sequence was discovered and predicted to be a secreted protein. Thenovel polypeptide encoded by the full length cDNA enable theidentification of a homolog relationship with adipocyte complementrelated protein Acrp30 (SEQ ID NO:8) and adipocyte secreted protein apM1(HUMUPST2_(—)1 in FIGS. 1 and 2, SEQ ID NO:3) . Somewhat more distanthomology was also identified to complement component Clq A chain, twofactors observed in the active state of hibernating Siberian woodchucks(HP25_TAMAS (SEQ ID NO:5) and HP27_TAMAS (SEQ ID NO:6)) and a rat brainprotein (CERL_RAT, SEQ ID NO:7), as shown in FIGS. 1 and 2.

[0063] The full sequence of the zsig39 polypeptide was obtained from asingle clone believed to contain it, wherein the clone was obtained froma lung tissue library. Other libraries that might also be searched forsuch clones include heart, small intestine, pancreas, skeletal muscle,kidney, thyroid, subthalamic nucleus, hippocampus, medulla oblongata,thalamus and the like.

[0064] The nucleotide sequence of the N-terminal EST is described in SEQID NO:1, and its deduced amino acid sequence is described in SEQ IDNO:2. As described generally above, the zsig39 polypeptide includes asignal sequence, ranging from amino acid 1 (Met) to amino acid residue15 (Gly). An alternative signal sequence ranges from amino acid 1 (Met)to amino acid 18 (Pro). The mature polypeptide therefore ranges fromamino acid 16 (Ser) or 19 (Leu) to amino acid 243 (Ala). Within themature polypeptide, an N-terminal region of limited homology is found,ranging between amino acid residue 20 (Asp) and 29 (Pro), wherein thecysteine at position 28 may provide similar structure/function as thecysteine found at position 36 in HUMUPST2_(—)1 and in the N-terminalregion of HP25_TAMAS and HP27_TAMAS. In addition, a collagen domain isfound between amino acid 30 (Gly) and 95 (Ala), 96 (Gly), 97 (Glu) or 98(Cys). In the collagen domain, 9 perfect Gly-Xaa-Pro and 13 or 14imperfect Gly-Xaa-Xaa repeats are observed. Acrp30 contains 22 perfector imperfect repeats.

[0065] The zsig39 polypeptide also includes a carboxy-terminal globulardomain, ranging from about amino acid 98 (Cys) or 99 (Ser) to 243 (Ala).The globular domain of ACRP30 has been determined to have a 10 betastrand “jelly roll” topology (Shapiro and Scherer, Curr. Biol. 8:335-8,1998) and the zsig39 sequence as represented by SEQ ID NO:2 contains all10 beta-strands of this structure (amino acid residues 105-109, 128-130,136-139, 143-146, 164-171, 176-182, 187-200, 204-210 and 226-231 of SEQID NO:2). These strands have been designated “A”, “A′”, “B”, “B′”, “C”,“D”, “BE”, “F”, “G” and “H” respectively. Also, two receptor bindingloops, amino acid residues 111-139 and 170-182 of SEQ ID NO:2, arerepresented. The core receptor binding region is predicted to includeamino acid residues 111-135 and 170-174 of SEQ ID NO:2. Those skilled inthe art will recognize that these boundaries are approximate, and arebased on alignments with known proteins and predictions of proteinfolding. Amino acid residues 149 (Glu), 151 (Tyr), 199 (Leu) and 227(Phe) appear to be conserved across the superfamily including CD40,TNFα, ACRP30 and zsig39.

[0066] The proteins of the present invention comprise a sequence ofamino acid residues that is at least 80% identical to SEQ ID NO:2.Within certain embodiments of the invention, the sequence is at least90% or 95% identical to SEQ ID NO:2.

[0067] Another aspect of the present invention includes zsig39polypeptide fragments. Preferred fragments include the collagen-likedomain of zsig39 polypeptides, ranging from amino acid 30 (Gly) to aminoacid-95 (Ala), 96 (Gly), 97 (Glu) or 98 (Cys) of SEQ ID NO:2, a portionof the zsig39 polypeptide containing the collagen-like domain or aportion of the collagen-like domain capable of dimerization oroligomerization. These fragments are particularly useful in the study ofcollagen dimerization or oligomerization or in formation of fusionproteins as described more fully below. Polynucleotides encoding suchfragments are also encompassed by the present invention, including thegroup consisting of (a) polynucleotide molecules comprising a sequenceof nucleotides as shown in SEQ ID NO: 1 from nucleotide 1, 198, 242, 251or 285 to nucleotide 482, 485, 488 or 491; (b) polynucleotide moleculesthat encode a zsig39 polypeptide fragment that is at least 80% identicalto the amino acid sequence of SEQ ID NO: 2 from amino acid residue 30(Gly) to amino-acid residue 96 (Gly), 97 (Glu), 98 (Cys); (c) moleculescomplementary to (a) or (b); and (f) degenerate nucleotide sequencesencoding a zsig39 polypeptide collagen-like domain fragment.

[0068] Such fragments or proteins containing such collagen-like domainsmay form homomeric constructs (dimers or oligomers of the same fragmentor protein). Moreover, such fragments or proteins containing suchcollagen-like domains may form heteromeric constructs (dimers oroligomers of different fragments or proteins). Other components ofheteromeric constructs may include Acrp30 and other polypeptidescharacterized by collagen-like domains as are described herein or knownin the art. These homomeric and heteromeric constructs are contemplatedby the present invention.

[0069] Other preferred fragments include the globular domain of zsig39polypeptides, ranging from amino acid 98(Cys) or 99 (Ser) to 243 (Ala)of SEQ ID NO:2, particularly from amino acid residue 105 to 231 of SEQID NO:2, a portion of the zsig39 polypeptide containing theglobular-like domain or an active portion of the globular-like domain.These fragments are particularly useful in the study or modulation ofenergy balance or neurotransmission, particularly diet- orstress-related neurotransmission. Anti-microbial activity may also bepresent in such fragments. The globular domain of Acrp30 proteins havebeen shown to assemble as a multimer of trimers. The trimers can be homoor heteromeric (Shapiro and Scherer, ibid.). Such fragments would alsobe useful for studying multimerization and receptor binding of zsig39and other related proteins such as Acrp30 and TNFα. Polynucleotidesencoding such fragments are also encompassed by the present invention,including the group consisting of (a) polynucleotide moleculescomprising a sequence of nucleotides as shown in SEQ ID NO:1 fromnucleotide 489 or 492 to nucleotide 926 or 1347; (b) polynucleotidemolecules that encode a zsig39 polypeptide fragment that is at least 800identical to the amino acid sequence of SEQ ID NO:2 from amino acidresidue 98 (Cys) or 99 (Ser) to amino acid residue 243 (Ala); (c)molecules complementary to (a) or (b); and (f) degenerate nucleotidesequences encoding a zsig39 polypeptide globular domain fragment.

[0070] Another zsig39 polypeptide fragment of the present inventioninclude both the collagen-like domain and the globular domain rangingfrom amino acid residue 30 (Gly) to 243 (Ala) of SEQ ID NO:2.Polynucleotides encoding such fragments are also encompassed by thepresent invention, including the group consisting of (a) polynucleotidemolecules comprising a sequence of nucleotides as shown in SEQ ID NO:1from nucleotide 285 to nucleotide 926 or 1347; (b) polynucleotidemolecules that encode a zsig39 polypeptide fragment that is at least80%, identical to the amino acid sequence of SEQ ID NO:2 from amino acidresidue 30 (Gly) to amino acid residue 243 (Ala); (c) moleculescomplementary to (a) or (b); and (f) degenerate nucleotide sequencesencoding a zsig39 polypeptide collagen-like domain-globular domainfragment.

[0071] Zsig39 fragments may be evaluated with respect to theiranti-microbial properties according to procedures known in the art. See,for example, Barsum et al., Eur. Respir. J. 8(5): 709-14, 1995;Sandovsky-Losica et al., J. Med. Vet. Mycol (England) 28(4): 279-87,1990; Mehentee et al., J. Gen. Microbiol (England) 135 (Pt. 8): 2181-8,1989; Segal and Savage, Journal of Medical and Veterinary Mycology 24:477-479, 1986 and the like. If desired, zsig39 polypeptide fragmentperformance in this regard can be compared to proteins known to befunctional in this regard, such as proline-rich proteins, lysozyme,histatins, lactoperoxidase or the like. In addition, zsig39 polypeptide-fragments may be evaluated in combination with one or moreanti-microbial agents to identify synergistic effects. One of ordinaryskill in the art will recognize that the anti-microbial properties ofzsig39 polypeptides, fusion proteins, agonists, antagonists andantibodies may be similarly evaluated.

[0072] As neurotransmitters or neurotransmission modulators, zsig39polypeptide fragments as well as zsig39 polypeptides, fusion proteins,agonists, antagonists or antibodies of the present invention may alsomodulate calcium ion concentration, muscle contraction, hormonesecretion, DNA synthesis or cell growth, inositol phosphate turnover,arachidonate release, phospholipase-C activation, gastric emptying,human neutrophil activation or ADCC capability, superoxide anionproduction and the like. Evaluation of these properties can be conductedby known methods, such as those set forth herein.

[0073] The impact of zsig39 polypeptide, fragment, fusion, agonist orantagonist on intracellular calcium level may be assessed by methodsknown in the art, such as those described by Dobrzanski et al.,Regulatory Peptides 45: 341-52, 1993, and the like. The impact of zsig39polypeptide, fragment, fusion, agonist or antagonist on musclecontraction may be assessed by methods known in the art, such as thosedescribed by Smits & Lebebvre, J. Auton. Pharmacol. 14: 383-92, 1994,Belloli et al., J. Vet. Pharmacol. Therap. 17: 379-83, 1994, Maggi etal., Regulatory Peptides 53: 259-74, 1994, and the like. The impact ofzsig39 polypeptide, fragment, fusion, agonist or antagonist on hormonesecretion may be assessed by methods known in the art, such as those forprolactin release described by Henriksen et al., J. of Receptor & SignalTransduction Research 15(1-4): 529-41, 1995, and the like. The impact ofzsig39 polypeptide, fragment, fusion, agonist or antagonist on DNAsynthesis or cell growth may be assessed by methods known in the art,such as those described by Dobrzanski et al., Regulatory Peptides 45:341-52, 1993, and the like. The impact of zsig39 polypeptide, fragment,fusion, agonist or antagonist on inositol phosphate turnover may beassessed by methods known in the art, such as those described byDobrzanski et al., Regulatory Peptides 45: 341-52, 1993, and the like.

[0074] Also, the impact of zsig39 polypeptide, fragment, fusion, agonistor antagonist on arachidonate release may be assessed by methods knownin the art, such as those described by Dobrzanski et al., RegulatoryPeptides 45: 341-52, 1993, and the like. The impact of zsig39polypeptide, fragment, fusion, agonist or antagonist on phospholipase-Cactivation may be assessed by methods known in the art, such as thosedescribed by Dobrzanski et al., Regulatory Peptides 45: 341-52, 1993,and the like. The impact of zsig39 polypeptide, fragment, fusion,agonist or antagonist on gastric emptying may be assessed by methodsknown in the art, such as those described by Varga et al., Eur. J.Pharmacol. 286: 109-112, 1995, and the like. The impact of zsig39polypeptide, fragment, fusion, agonist or antagonist on human neutrophilactivation and ADCC capability may be assessed by methods known in theart, such as those described by Wozniak et al., Immunology 78: 629-34,1993, and the like. The impact of zsig39 polypeptide, fragment, fusion,agonist or antagonist on superoxide anion production may be assessed bymethods known in the art, such as those described by Wozniak et al.,Immunology 78: 629-34, 1993, and the like.

[0075] The present invention also provides zsig39 fusion proteins. Forexample, fusion proteins of the present invention encompass (1) apolypeptide selected from the following: a) a polypeptide comprising asequence of amino acid residues that is at least 80% identical in aminoacid sequence to amino acid residue 19 to amino acid residue 243 of SEQID NO:2; b) a polypeptide comprising a sequence of amino acid residuesas shown in SEQ ID NO:2 from amino acid residue 16 to amino acid residue243; c) a polypeptide comprising a sequence of amino acid residues asshown in SEQ ID NO:2 from amino acid residue 1 to amino acid residue243; d) a portion of the zsig39 polypeptide as shown in SEQ ID NO:2containing the collagen-like domain or a portion of the collagen-likedomain capable of dimerization or oligomerization; e) a portion of thezsig39 polypeptide as shown in SEQ ID NO:2, containing the globular-likedomain or an active portion of the globular-like domain; or f) a portionof the zsig39 polypeptide as shown in SEQ ID NO:2, including thecollagen-like domain and the globular domain; and (2) anotherpolypeptide. The other polypeptide may be alternative or additionalglobular domain, an alternative or additional collagen-like domain, asignal peptide to facilitate secretion of the fusion protein or thelike. The globular domain of complement bind IgG, thus, the globulardomain of zsig39 polypeptide, fragment or fusion may have a similarrole.

[0076] Zsig39 polypeptides, ranging from amino acid 1 (Met) to aminoacid 243 (Ala); the alternative mature zsig39 polypeptides, ranging fromamino acid 16 (Ser) or amino acid 19 (Leu) to amino acid 243 (Ala); orthe alternative secretion leader fragments thereof, which fragmentsrange from amino acid 1 (Met) to amino acid 15 (Gly) or amino acid 18(Pro) may be used in the study of secretion of proteins from cells. Inpreferred embodiments of this aspect of the present invention, themature polypeptides are formed as fusion proteins with putativesecretory signal sequences; plasmids bearing regulatory regions capableof directing the expression of the fusion protein is introduced intotest cells; and secretion of mature protein is monitored. In otherpreferred embodiments of this aspect of the present invention, thealternative secretion leader fragments are formed as fusion proteinswith alternative proteins selected for secretion; plasmids bearingregulatory regions capable of directing the expression of the fusionprotein are introduced into test cells; and secretion of the protein ismonitored. The monitoring may be done by techniques known in the art,such as HPLC and the like.

[0077] The highly conserved amino acids, particularly those in thecarboxy-terminal globular domain of zsig39 polypeptide, can be used as atool to identify new family members. For instance, reversetranscription-polymerase chain reaction (RT-PCR) can be used to amplifysequences encoding the conserved motifs from RNA obtained from a varietyof tissue sources: In particular, highly degenerate primers designedfrom conserved sequences are useful for this purpose. In particular, thefollowing primers are useful for this purpose:

[0078] 1) Amino acids 121-126 of SEQ ID NO: 2 (corresponding tonucleotides 558-575 of SEQ ID NO: 1);

[0079] 2) Amino acids 131-136 of SEQ ID NO: 2 (corresponding tonucleotides 588-605 of SEQ ID NO: 1);

[0080] 3) Amino acids 149-154 of SEQ ID NO: 2 (corresponding tonucleotides 642-659 of SEQ ID NO: 1);

[0081] 4) Amino acids 202-207 of SEQ ID NO: 2 (corresponding tonucleotides 801-818 of SEQ ID NO: 1); and

[0082] 5) Amino acids 226-231 of SEQ ID NO: 2 (corresponding tonucleotides 873-890 of SEQ ID NO: 1).

[0083] The present invention also contemplates degenerate probes basedupon the polynucleotides described above. Probes corresponding tocomplements of the polynucleotides set forth above are also encompassed.

[0084] Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NO: 2,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, other probes specificallyrecited herein or a sequence complementary thereto, under stringentconditions. In general, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Typicalstringent conditions are those in which the salt concentration is up toabout 0.03 M at pH 7 and the temperature is at least about 60° C.

[0085] Those skilled in the art will readily recognize that, in view ofthe degeneracy of the genetic code, considerable sequence variation ispossible among these polynucleotide molecules. SEQ ID NO:10 is adegenerate polynucleotide sequence that encompasses all polynucleotidesthat could encode the zsig39 polypeptide of SEQ ID NO:2 (amino acids1-243). Those skilled in the art will also recognize that the degeneratesequence of SEQ ID NO:10 also provides all RNA sequences encoding SEQ IDNO:2 by substituting U for T. Thus, zsig39 polypeptide-encodingpolynucleotides ranging from nucleotide 1, 46 or 55 to nucleotide 729 ofSEQ ID NO:10 are contemplated by the present invention. Alsocontemplated by the present invention are fragments and fusions asdescribed above with respect to SEQ ID NO:1, which are formed fromanalogous regions of SEQ ID NO:10, wherein nucleotides 198 to 926 of SEQID NO:1 correspond to nucleotides 1 to 729 of SEQ ID NO:10. The symbolsin SEQ ID NQ:10 are summarized in Table 1 below. TABLE 1 NucleotideResolutions Complement Resolutions A A T T C C G G G G C C T T A A R A|GY C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G W A|T W A|T S C|G HA|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|TN A|C|G|T

[0086] The degenerate codons used in SEQ ID NO:10, encompassing allpossible codons for a given amino acid, are set forth in Table 2 below.TABLE 2 Amino Degenerate Acid Letter Codons Codon Cys C TGC TGT TGY SerS AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT CAN Pro P CCA CCCCCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN Asn NAAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CAR His HCAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AAR Met MATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val VGTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGG TGGTer . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN Gap - . . .

[0087] One of ordinary skill in the art will appreciate that someambiguity is introduced in determining a degenerate codon,representative of all possible codons encoding each amino acid. Forexample, the degenerate codon for serine (WSN) can, in somecircumstances, encode arginine (AGR), and the degenerate codon forarginine (MGN) can, in some circumstances, encode serine (AGY). Asimilar relationship exists between codons encoding phenylalanine andleucine. Thus, some polynucleotides encompassed by the degeneratesequence may have some incorrect amino acids, but one of ordinary skillin the art can easily identify such erroneous sequences by reference tothe amino acid sequence of SEQ ID NO: 2.

[0088] Within another aspect of the present invention there is provideda pharmaceutical composition comprising purified zsig39 polypeptide incombination with a pharmaceutically acceptable vehicle. Thispharmaceutical composition will be used to modulate energy balance inmammals or to protect endothelial cells from injury.

[0089] The expression pattern of zsig39 polypeptide indicates expressionin endothelial cell tissues. With regard to endothelial cell protection,zsig39 polypeptide may be used in organ preservation, forcryopreservation, for surgical pretreatment to prevent injury due toischemia and/or inflammation or in like procedures. The high expressionlevel in the small intestine suggests that zsig39 polypeptide may be anendogenous factor that protects gastrointestinal tissue from ischemicreperfusion injury. Rat, rabbit and pig models of ischemic reperfusioninjury are known in the art and may be used to evaluate zsig39, agonistsor antagonists thereof, antibodies, fusion proteins and fragments. Forexample, Golino et al., Nature Medicine, 2(1): 35-40, 1996, describe amyocardial model of ischemic reperfusion injury employing New Zealandwhite rabbits. New Zealand white rabbits have also been employed in (1)an ischemic reperfusion model of the central vein in the ear and (2) aatherosclerotic femoral artery injury model in which blood flow isreinstated by balloon angioplasty. See, for example, Winn et al., J.Clin. Invest., 92: 2042-7, 1993, and Jang et al., Circulation, 92(10):3041-50, 1995.

[0090] A rat model of gut ischemia may also be employed. For example,male Sprague Dawley rats weighing between 225 and 400 grams undergothree training sessions with regard to sitting quietly in restrainingcages. Next, the rats undergo a survival surgery, during which jugularvein catheters are implanted. For the survival surgery, rats areanesthetized, and catheter is implanted in the right jugular vein underconditions selected to maintain patency. The rats are then placed inrestrainer cages and receive administrations of the test composition orvehicle as described below.. The rats were allowed to recover for 48hours prior to a 4 day single intravenous bolus injection (0.5 ml) perday of either vehicle or test composition. The rats are fasted,preferably for 16-24 hours, anesthetized, and given an analgesic, priorto the fourth injection. Thirty minutes after the fourth injection, theabdomen of each rat is opened with a small incision, and the superiormesenteric artery is isolated and clamped for one hour. The abdomen isloosely sutured closed during the clamping period, reopened for removalof the clamp and again loosely sutured closed. The rats are placed intoholding cages resting on a 37° C. heating pad for a two hour reperfusionperiod. Following the reperfusion period, the rats are sacrificed andjejunal intestinal segments are excised. Some excised intestinalsegments are subject to histological evaluation and others are analyzedfor myeloperoxidase (MPO) and maltase activities.

[0091] MPO is a measure of the amount of neutrophil infiltration intothe tissue, while maltase activity is a measure of the integrity of theintestinal mucosa. Ischemic reperfusion injury is associated withincreased levels of MPO and reduced levels of maltase activity.Consequently, amelioration of ischemic reperfusion injury is expected toresult in reduced MPO and increased maltase activity.

[0092] Also, zsig39 polypeptide is expressed in the subthalamic nucleus,suggesting that zsig39 polypeptide or agonist thereof may be anendogenous suppressor of ballistic movement by delivering an inhibitorystimulus to chronically active cells. Such ballistic movements resultfrom lesion of subthalamic nuclei. Evaluation of zsig39 polypeptide,agonists or antagonists thereof, antibodies, fusion proteins andfragments for efficacy in suppressing ballistic movements may beconducted using techniques that are known in the art. For example,stereotactic instruments can be used to lesion the subthalamic nuclei;if ballistic movement is observed, zsig39 polypeptide, agonists orantagonists thereof, antibodies, fusion proteins or fragments areadministered; and any modulation of ballistic movement is noted.

[0093] With regard to modulating energy balance, zsig39 polypeptidesmodulate cellular metabolic reactions. Such metabolic reactions includeadipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucoseuptake, protein synthesis, thermogenesis oxygen utilization and thelike. Among other methods known in the art or described herein,mammalian energy balance may be evaluated by monitoring one or more ofthe aforementioned metabolic functions. These metabolic functions aremonitored by techniques (assays or animal models) known to one ofordinary skill in the art, as is more fully set forth below. Forexample, the glucoregulatory effects of insulin are predominantlyexerted in the liver, skeletal muscle and adipose tissue. Insulin bindsto its cellular receptor in these three tissues and initiatestissue-specific actions that result in, for example, the inhibition ofglucose production and the stimulation of glucose utilization. In theliver, insulin stimulates glucose uptake and inhibits gluconeogeriesisand glycogenolysis. In skeletal muscle and adipose tissue, insulin actsto stimulate the uptake, storage and utilization of glucose.

[0094] Art-recognized methods exist for monitoring all of the metabolicfunctions recited above. Thus, one of ordinary skill in the art is ableto evaluate zsig39 polypeptides, fragments, fusion proteins, antibodies,agonists and antagonists for metabolic modulating functions. Exemplarymodulating techniques are set forth below.

[0095] Adipogenesis, gluconeogenesis and glycogenolysis are interrelatedcomponents of mammalian energy balance, which may be evaluated by knowntechniques using, for example, ob/ob mice or db/db mice. The ob/ob miceare inbred mice that are homozygous for an inactivating mutation at theob (obese) locus. Such ob/ob mice are hyperphagic and hypometabolic, andare believed to be deficient in production of circulating OB protein.The db/db mice are inbred mice that are homozygous for an inactivatingmutation at the ob (diabetes) locus. The db/db mice display a phenotypesimilar to that of ob/ob mice, except db/db mice also display a diabeticphenotype. Such db/db mice are believed to be resistant to the effectsof circulating OB protein. Also, various in vitro methods of assessingthese parameters are known in the art.

[0096] Insulin-stimulated lipogenesis, for example, may be monitored bymeasuring the incorporation of ¹⁴C-acetate into triglyceride (Mackall etal. J. Biol. Chem. 251:6462-6464, 1976) or triglyceride accumulation(Kletzien et al., Mol. Pharmacol. 41:393-398, 1992).

[0097] Glucose uptake may be evaluated, for example, in an assay forinsulin-stimulated glucose transport. Non-transfected, differentiated L6myotubes (maintained in the absence of G418) are placed in DMEMcontaining 1 g/l glucose, 0.5 or 1.00 BSA, 20 mM Hepes, and 2 mMglutamine. After two to five hours of culture, the medium is replacedwith fresh, glucose-free DMEM containing 0.5 or 1.0% BSA, 20 mM Hepes, 1mM pyruvate, and 2 mM glutamine. Appropriate concentrations of insulinor IGF-1, or a dilution series of the test substance, are added, and thecells are incubated for 20-30 minutes. ³H or ¹⁴C-labeled deoxyglucose isadded to ≈50 1 M final concentration, and the cells are incubated forapproximately 10-30 minutes. The cells are then quickly rinsed with coldbuffer (e.g. PBS), then lysed with a suitable lysing agent (e.g. 1 SDSor 1 N NaOH). The cell lysate is then evaluated by counting in ascintillation counter. Cell-associated radioactivity is taken as ameasure of glucose transport after subtracting non-specific binding asdetermined by incubating, cells in the presence of cytocholasin b, aninhibitor of glucose transport. Other methods include those describedby, for example, Manchester et al., Am. J. Physiol. 266 (Endocrinol.Metab. 29):E326-E333, 1994 (insulin-stimulated glucose transport).

[0098] Fatty acid metabolism may also be monitored by techniques knownin the art. In particular, uptake and metabolism of fatty acids by theheart. Suitable animal models are available and tissues are available.Cultured cells include cardiac fibroblasts and cardiac myocytes.Established cell lines include: NIH 3T3 fibroblast (ATCC No. CRL-1658),CHH-1 chum heart cells (ATCC No. CRL-1680) and H9c2 rat heart myoblasts(ATCC No. CRL-1446). It has been demonstrated that as cardiac cells agethere is a shift from fatty acid metabolism to glucose metabolism (Sacket al., Circulation 94:2837-42, 1996).

[0099] Protein synthesis may be evaluated, for example, by comparingprecipitation of ³⁵S-methionine-labeled proteins following incubation ofthe test cells with ³⁵S-methionine and ³⁵S-methionine and a putativemodulator of protein synthesis.

[0100] Thermogenesis may be evaluated as described by B. Stanley in TheBiology of Neuropeptide Y and Related Peptides, W. Colmers and C.Wahlestedt (eds.), Humana Press, Ottawa, 1993, pp. 457-509; C.Billington et al., Am. J. Physiol. 260:R321, 1991; N. Zarjevski et al.,Endocrinolocy 133:1753, 1993; C. Billington et al., Am. J. Physiol.266:R1765, 1994; Heller et al., Am. J. Physiol. 252(4 Pt 2): R661-7,1987; and Heller et al., Am. J. Physiol. 245(3): R321-8, 1983. Also,metabolic rate, which may be measured by a variety of techniques, is anindirect measurement of thermogenesis.

[0101] Oxygen utilization may be evaluated as described by Heller etal., Pflugers Arch 369(1): 55-9, 1977. This method also involved ananalysis of hypothalmic temperature and metabolic -heat production.Oxygen utilization and thermoregulation have also been evaluated inhumans as described by Haskell et al., J. Appl. Physiol. 51(4): 948-54,1981.

[0102] Expression of zsig39 polypeptide in the heart and in brain tissueinvolved in involuntary function (i.e., the medulla oblongata) suggeststhat the protein may modulate acetylcholine and/or norepinephrinerelease. Among other methods known in the art or described herein,mammalian endothelial cell tissue protection may be evaluated bymonitoring the function of endothelial tissue. For example, the functionof the heart (aorta) may be evaluated by monitoring acetylcholinerelease, norepinephrine release or like parameters. These parameters aremonitored by techniques (assays or animal models) known to one ofordinary skill in the art, as is more fully set forth below.

[0103] Acetylcholine and norepinephrine release may be monitored byHPLC. Levy, Electrophysiology of the Sinoatrial and AtrioventricularNodes, Alan R. Liss, Inc., 187-197, 1998, describe measurement ofnorepinephrine in coronary sinus effluent. In addition, animals may beelectrically paced, with the results monitored as described by Elsner,European Heart Journal 16(Supplement N) 52-8, 1995, and Reiffel andKuehnert, PACE 17(Part 1): 349-65, 1994.

[0104] Zsig39 polypeptides may also find use as neurotransmitters or asmodulators of neurotransmission, as indicated by expression of thepolypeptide in tissues associated with the sympathetic orparasympathetic nervous system. In this regard, zsig39 polypeptides mayfind utility in modulating nutrient uptake, as demonstrated, forexample, by 2-deoxy-glucose uptake in the brain or the like.

[0105] Among other methods known in the art or described herein,neurotransmission functions may be evaluated by monitoring2-deoxy-glucose uptake in the brain. This parameter is monitored bytechniques (assays or animal models) known to one of ordinary skill inthe art, for example, autoradiography. Useful monitoring techniques aredescribed, for example, by Kilduff et al., J. Neurosci. 10 2463-75,1990, with related techniques used to evaluate the “hibernating heart”as described in Gerber et al. Circulation 94(4): 651-8, 1996, andFallavollita et al., Circulation 95(7): 1900-1909, 1997.

[0106] In addition, zsig39 polypeptides, fragments, fusions agonists orantagonists thereof may be therapeutically useful for anti-microbial orneurotransmitter-modulated applications. For example, complementcomponent Clq plays a role in host defense against infectious agents,such as bacteria and viruses. Clq is known to exhibit severalspecialized functions. For example, Clq triggers the complement cascadevia interaction with bound antibody or C-reactive protein (CRP). Also,Clq interacts directly with certain bacteria, RNA viruses, mycoplasma,uric acid crystals, the lipid A component of bacterial endotoxin andmembranes of certain ihtracellular organelles. Clq binding to the Clqreceptor is believed to promote phagocytosis. Clq also appears toenhance the antibody formation aspect of the host defense system. See,for example, Johnston, Pediatr. Infect. Dis. J. 12(11): 933-41, 1993.Thus, soluble Clq-like molecules may be useful as anti-microbial agents,promoting lysis or phagocytosis of infectious agents.

[0107] The zsig39 polypeptides of the present invention also exhibithomology to moieties believed to modulate neurotransmission. As shown inFIG. 1, zsig39 polypeptides are homologous to the following proteins:HP25_TAMAS (SEQ ID NO:5) (Takamatsu et al., Mol. Cell. Biol. 13:1516-21, 1993 and Kondo & Kondo, J. Biol. Chem. 267: 473-8, 1992);HP27_TAMAS (SEQ ID NO:6) (Takamatsu et al. and Kondo & Kondo referencedabove) and CERL_RAT (SEQ ID NO:7) (Wada & Ohtani, Brain Res. Mol. BrainRes. 9: 71-7, 1991). HP25 and HP27 are polypeptides found in the active(summer) serum of hibernating Siberian woodchucks. CERL is present inthe rat cerebellum. Thus, zsig39 polypeptides, fragments fusions,agonists or antagonists may be useful in modulating neurotransmissionby, for example, binding to neurotransmitters or receptors therefor.

[0108] Radiation hybrid mapping is a somatic cell genetic techniquedeveloped for constructing high-resolution, contiguous maps of mammalianchromosomes (Cox et al., Science 250:245-250, 1990). Partial or fullknowledge of a gene's sequence allows the designing of PCR primerssuitable for use with chromosomal radiation hybrid mapping panels.Commercially available radiation hybrid mapping panels which cover theentire human genome, such as the Stanford G3 RH Panel and the GeneBridge4 RH Panel (Research Genetics, Inc., Huntsville, Ala.), are available.These panels enable rapid, PCR based, chromosomal localizations andordering of genes, sequence-tagged sites (STSs), and othernonpolymorphic- and polymorphic markers within a region of interest.This includes establishing directly proportional physical distancesbetween newly discovered genes of interest and previously mappedmarkers. The precise knowledge of a genets position can be useful in anumber of ways including: 1) determining if a sequence is part of anexisting contig and obtaining additional surrounding genetic sequencesin various forms such as YAC-, BAC- or cDNA clones, 2) providing apossible candidate gene for an inheritable disease which shows linkageto the same chromosomal region, and 3) for cross-referencing modelorganisms such as mouse which may be beneficial in helping to determinewhat function a particular gene might have.

[0109] The results showed that the zsig39 polypeptide-encoding gene maps549.99 cR_(—)3000 from the top of the human chromosome 11 linkage groupon the WICGR radiation hybrid map-. Proximal and distal frameworkmarkers were AFMB048ZA9 and FB17D4, respectively. The use of surroundingmarkers positions the zsig39 gene in the 11q23.3 region on theintegrated LDB chromosome 11 map (The Genetic Location Database,University of Southhampton, WWW server:http://cedar.genetics.soton.ac.uk/public_html/).

[0110] The present invention also provides reagents which will find usein diagnostic applications. For example, the zsig39 gene, a probecomprising zsig39 DNA or RNA or a subsequence thereof can be used todetermine if the zsig39 gene is present on chromosome 11 or if amutation has occurred. Detectable chromosomal aberrations at the zsig39gene locus include but are not limited to aneuploidy, gene copy numberchanges, insertions, deletions, restriction site changes andrearrangements.

[0111] In general, these diagnostic methods comprise the steps of (a)obtaining a genetic sample from a patient; (b) incubating the geneticsample with a polynucleotide probe or primer as disclosed above, underconditions wherein the polynucleotide will hybridize to complementarypolynucleotide sequence, to produce a first reaction product; and (iii)comparing the first reaction product to a control reaction product. Adifference between the first reaction product and the control reactionproduct is indicative of a genetic abnormality in the patient. Geneticsamples for use within the present invention include genomic DNA, cDNA,and RNA. The polynucleotide probe or primer can be RNA or DNA, and willcomprise a portion of SEQ ID NO:1, the complement of SEQ ID NO:1, or anRNA equivalent thereof. Suitable assay methods in this regard includemolecular genetic techniques known to those in the art, such asrestriction fragment length polymorphism (RFLP) analysis, short tandemrepeat (STR) analysis employing PCR techniques, ligation chain reaction(Barany, PCR Methods and Applications 1:5-16, 1991); ribonucleaseprotection assays, and other genetic linkage analysis techniques knownin the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian,Chest 108:255-65, 1995). Ribonuclease protection assays (see, e.g.,Ausubel et al., ibid., ch. 4) comprise the hybridization of an RNA probeto a patient RNA sample, after which the reaction product (RNA-RNAhybrid) is exposed to RNase. Hybridized regions of the RNA are protectedfrom digestion. Within PCR assays, a patient's genetic sample isincubated with a pair of polynucleotide primers, and the region betweenthe primers is amplified and recovered. Changes in size or amount ofrecovered product are indicative of mutations in the patient. AnotherPCR-based technique that can be employed is single strand conformationalpolymorphism (SSCP) analysis (Hayashi, PCR Methods and Applications1:34-8, 1991).

[0112] Zsig39 polypeptides may be used in the analysis of energyefficiency of a mammal. Zsig39 polypeptides found in serum or tissuesamples may be indicative of a mammals ability to store food, with morehighly efficient mammals tending toward obesity. More specifically, thepresent invention contemplates methods for detecting zsig39 polypeptidecomprising:

[0113] exposing a sample possibly containing zsig39 polypeptide to anantibody attached to a solid support, wherein said antibody binds to anepitope of a zsig39 polypeptide;

[0114] washing said immobilized antibody-polypeptide to remove unboundcontaminants;

[0115] exposing the immobilized antibody-polypeptide to a secondantibody directed to a second epitope of a zsig39 polypeptide, whereinthe second antibody is associated with a detectable label; and

[0116] detecting the detectable label. The concentration of zsig39polypeptide in the test sample appears to be indicative of the energyefficiency of a mammal. This information can- aid nutritional analysisof a mammal. Potentially, this information may be useful in identifyingand/or targeting energy deficient tissue.

[0117] As is described in greater detail below, mice receiving zsig39were found to have decreased levels of serum free fatty acids and aincrease in bone fat. Fatty acids are incorporated into triglyceridesand stored as fat. The stored fat acts to insulate the body from heatloss and protect internal organs. Fat also serves as a repository ofstored energy. Fatty acids are released from the triglycerides byhormone-regulated lipases for use in energy metabolism. Decrease in freefatty acid levels suggests zsig39 has an effect on the uptake andmetabolism of free fatty acids. Zsig39 may act to inhibit the of releaseof fatty acids from fat reserves, such as by inhibiting the action ofhormonal lipases. Zsig39 may also act to enhance fatty acid uptake,metabolism and storage. Zsig39 may act independently or in concert withother molecules, such as insulin, to inhibit lipolysis, enhance fattyacid uptake and/or metabolism. As such, zsig39 would be useful inregulation of energy metabolism. The invention therefore provides amethod for modulating free fatty acid metabolism in individuals in needof such treatment by administering to such an individual apharmaceutically effective dose of a zsig39 polypeptide. A“pharmaceutically effective amount” of a zsig39 polypeptide is an amountsufficient to induce a desired biological result. The result can bealleviation of the signs, symptoms, or causes of a disease, or any otherdesired alteration of a biological system. For example, an effectiveamount of a zsig39 polypeptide, agonist or antagonist is that whichprovides either subjective relief of symptoms or an objectivelyidentifiable improvement as noted by the clinician or other qualifiedobserver. In particular, such an effective amount of a zsig39polypeptide results in reduction serum free fatty acid levels or otherbeneficial effect. Effective amounts of the zsig39 polypeptides can varywidely depending on the disease or symptom to be treated. The amount ofthe polypeptide to be administered, and its concentration in theformulations, depends upon the vehicle selected, route ofadministration, the potency of the particular polypeptide, the clinicalcondition of the patient, the side effects and the stability of thecompound in the formulation. Thus, the clinician will employ theappropriate preparation containing the appropriate concentration in theformulation, as well as the amount of formulation administered,depending upon clinical experience with the patient in question or withsimilar patients. Such amounts will depend, in part, on the particularcondition to be treated, age, weight, and general health of the patient,and other factors evident to those skilled in the art.

[0118] Within additional aspects of the invention there are providedantibodies or synthesized binding proteins (e.g., those generated byphage display, E. coli Fab, and the like) that specifically bind to thezsig39 polypeptides described above. Such antibodies are useful for,among other uses as described herein, preparation of anti-idiotypicantibodies. Synthesized binding proteins may be produced by phagedisplay using commercially available kits, such as the Ph.D.™ PhageDisplay Peptide Library Kits available from New England Biolabs, Inc.(Beverly, Mass.). Phage display techniques are described, for example,in U.S. Pat. Nos. 5,223,409, 5,403,484 and 5,571,698.

[0119] An additional aspect of the present invention provides methodsfor identifying agonists or antagonists of the zsig39 polypeptidesdisclosed above, which agonists or antagonists may have valuableproperties as discussed further herein. Within one embodiment, there isprovided a method of identifying zsig39 polypeptide agonists, comprisingproviding cells responsive thereto, culturing the cells in the presenceof a test compound and comparing the cellular response with the cellcultured in the presence of the zsig39 polypeptide, and selecting thetest compounds for which the cellular response is of the same type.

[0120] Within another embodiment, there is provided a method ofidentifying antagonists of zsig39 polypeptide, comprising providingcells responsive to a zsig39 polypeptide, culturing a first portion ofthe cells in the presence of zsig39 polypeptide, culturing a secondportion of the cells in the presence of the zsig39 polypeptide and atest compound, and detecting a decrease in a cellular response of thesecond portion of the cells as compared to the first portion of thecells.

[0121] In addition to those assays disclosed herein, samples can betested for inhibition of zsig39 activity within a variety of assaysdesigned to measure receptor binding or the stimulation/inhibition ofzsig39-dependent cellular responses. For example, zsig39-responsive celllines can be transfected with a reporter gene construct that isresponsive to a zsig39-stimulated cellular pathway. Reporter geneconstructs of this type are known in the art, and will generallycomprise a zsig39-DNA response element operably linked to a geneencoding an assayable protein, such as luciferase DNA response elementscan include, but are not limited to, cyclic AMP response elements (CRE),hormone response elements (HRE) insulin response element (IRE) (Nasrinet al., Proc. Natl. Acad. Sci. USA 87:5273-7, 1990) and serum responseelements (SRE) (Shaw et al. Cell 56: 563-72, 1989). Cyclic AMP responseelements are reviewed in Roestler et al., J. Biol. Chem. 263(19):9063-6; 1988 and Habener, Molec. Endocrinol. 4 (8):1087-94; 1990.Hormone response elements are reviewed in Beato, Cell 56:335-44; 1989.Candidate compounds, solutions, mixtures or extracts are tested for theability to inhibit the activity of zsig39 on the target cells asevidenced by a decrease in zsig39 stimulation of reporter geneexpression. Assays of this type will detect compounds that directlyblock zsig39 binding to cell-surface receptors, as well as compoundsthat block processes in the cellular pathway subsequent toreceptor-ligand binding. In the alternative, compounds or other samplescan be tested for direct blocking of zsig39 binding to receptor usingzsig39 tagged with a detectable label (e.g., ¹²⁵I, biotin, horseradishperoxidase, FITC, and the like). Within assays of this type, the abilityof a test sample to inhibit the binding of labeled zsig39 to thereceptor is indicative of inhibitory activity, which can be confirmedthrough secondary assays. Receptors used within binding assays may becellular receptors or isolated, immobilized receptors.

[0122] A further aspect of the invention provides a method for studyinginsulin. Such methods of the-present invention comprise incubatingadipocytes in a culture medium comprising zsig39 polypeptide, monoclonalantibody, agonist or antagonist thereof ± insulin and observing changesin adipocyte protein secretion or differentiation.

[0123] Anti-microbial protective agents may be directly acting orindirectly acting. Such agents operating via membrane association orpore forming mechanisms of action directly attach to the offendingmicrobe. Anti-microbial agents can also act via an enzymatic mechanism,breaking down microbial protective substances or the cell wall/membranethereof. Anti-microbial agents, capable of inhibiting microorganismproliferation or action or of disrupting microorganism integrity byeither mechanism set forth above, are useful in methods for preventingcontamination in cell culture by microbes susceptible to thatanti-microbial activity. Such techniques involve culturing cells in thepresence of an effective amount of said zsig39 polypeptide or an agonistor antagonist thereof.

[0124] Also, zsig39 polypeptides or agonists thereof may be used as cellculture reagents in in vitro studies of exogenous microorganisminfection, such as bacterial, viral or fungal infection. Such moietiesmay also be used in in vivo animal models of infection.

[0125] The present invention also provides methods of studying mammaliancellular metabolism. Such methods of the present invention compriseincubating cells to be studies, for example, human vascular endothelialcells, ± zsig39 polypeptide, monoclonal antibody, agonist or antagonistthereof and observing changes in adipogenesis, gluconeogenesis,glycogenolysis, lipogenesis, glucose uptake, or the like.

[0126] An additional aspect of the invention provides a method forstudying dimerization or oligomerization. Such methods of the presentinvention comprise incubating zsig39 polypeptides or fragments or fusionproteins thereof containing a collagen-like domain alone or incombination with other polypeptides bearing collagen-like domains andobserving the associations formed between the collagen like domains.Thus, both homomeric and heteromeric constructs may be studied in thismanner. Such associations are indicated by HPLC, circular dichroism orthe like.

[0127] As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for isolating DNA and RNA arewell known in the art. It is generally preferred to isolate RNA frombrain tumor, heart, placenta, adipose tissue and the like, although DNAcan also be prepared using RNA from other tissues or isolated as genomicDNA. Total RNA can be prepared using guanidine HCl extraction followedby isolation by centrifugation in a CsCl gradient (Chirgwin et al.,Biochemistry 18:52-94, 1979). Poly (A)⁺ RNA is prepared from total RNAusing the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA69:1408-1412, 1972). Complementary DNA (cDNA) is prepared from poly(A)⁺RNA using known methods. Polynucleotides encoding zsig39 polypeptidesare then identified and isolated by, for example, hybridization or PCR.

[0128] The present invention further provides counterpart polypeptidesand polynucleotides from other species (orthologs). These speciesinclude, but are not limited to mammalian, avian, amphibian, reptile,fish, insect and other vertebrate and invertebrate species. Ofparticular interest are zsig39 polypeptides from other mammalianspecies, including murine, rat, porcine, ovine, bovine, canine, feline,equine and other primate proteins. Orthologs of the human proteins canbe cloned using information and compositions provided by the presentinvention in combination with conventional cloning techniques. Forexample, a cDNA can be cloned using mRNA obtained from a tissue or celltype that expresses the protein. Suitable sources of mRNA can beidentified by probing Northern blots with probes designed from thesequences disclosed herein. A library is then prepared from mRNA of apositive tissue of cell line. A zsig39 polypeptide-encoding cDNA canthen be isolated by a variety of methods, such as by probing with acomplete or partial human cDNA or with one or more sets of degenerateprobes based on the disclosed sequences. A cDNA can also be cloned usingthe polymerase chain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202),using primers designed from the sequences disclosed herein. Within anadditional method, the cDNA library can be used to transform ortransfect host cells, and expression of the cDNA of interest can bedetected with an antibody to zsig39 polypeptide. Similar techniques canalso be applied to the isolation of genomic clones.

[0129] Those skilled in the art will recognize that the sequencesdisclosed in SEQ ID NO:1 and SEQ ID NO:2 represent a single allele ofhuman zsig39 DNA and protein and. that allelic variation and alternativesplicing are expected to occur. Allelic variants of this sequence can becloned by probing cDNA or genomic libraries from different individualsaccording to standard procedures. Allelic variants of the DNA sequenceshown in SEQ ID NO:1, including those containing silent mutations andthose in which mutations result in amino acid sequence changes, arewithin the scope of the present invention, as are proteins which areallelic variants of SEQ ID NO:2. cDNAs generated from alternativelyspliced mRNAs, which retain the properties of the zsig39 polypeptide areincluded within the scope of the present invention, as are polypeptidesencoded by such cDNAs and mRNAs. Allelic variants and splice variants ofthese sequences can be cloned by probing cDNA or genomic libraries fromdifferent individuals or tissues according to standard procedures knownin the art.

[0130] The present invention also provides isolated zsig39 polypeptidesthat are substantially homologous to the polypeptides of SEQ ID NO:2 andtheir species orthologs. The term “substantially homologous” is usedherein to denote polypeptides having 50%, preferably 60%, morepreferably at least 80%, sequence identity to the sequences shown in SEQID NO:2 or their orthologs. Such polypeptides will more preferably be atleast 90% identical, and most preferably 95% or more identical to SEQ IDNO:2 or its orthologs. Percent sequence identity is determined byconventional methods. See, for example, Altschul et al., Bull. Math.Bio. 48: 603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci.USA 89:10915-10919, 1992. Briefly, two amino acid sequences are alignedto optimize the alignment scores using a gap opening penalty of 10, agap extension penalty of 1, and the “blosum 62” scoring matrix ofHenikoff and Henikoff (ibid.) as shown in Table 3 (amino acids areindicated by the standard one-letter codes). The percent identity isthen calculated as:

Total number of identical matches/[length of the longer sequence plusthe number of gaps introduced into the longer sequence in order to alignthe two sequences]×100 TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A4 R −1 5 N −2 0 6 D −2 −2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2−4 2 5 G 0 −2 0 −1 −3 −2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1−3 −3 −4 −3 4 L −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1−3 −2 5 M −1 −1 −2 −3 −1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3−1 0 0 −3 0 6 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0−1 0 0 0 −1 −2 −2 0 −1 −2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2−1 1 5 W −3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2−3 −2 −1 −2 −3 2 −1 −1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −33 1 −2 1 −1 −2 −2 0 −3 −1 4

[0131] Sequence identity of polynucleotide molecules is determined bysimilar methods using a ratio as disclosed above.

[0132] Substantially homologous proteins and polypeptides arecharacterized as having one or more amino acid substitutions, deletionsor additions. These changes are preferably of a minor nature, that isconservative amino acid substitutions (see Table 4) and othersubstitutions that do not significantly affect the folding or activityof the protein or polypeptide; small deletions, typically of one toabout 30 amino acids; and small amino- or carboxyl-terminal extensions,such as an amino-terminal methionine residue, a small linker peptide ofup to about 20-25 residues, or a small extension that facilitatespurification, an affinity tag. Polypeptides comprising affinity tags canfurther comprise a proteolytic cleavage site between the zsig39polypeptide and the affinity tag. Preferred such sites include thrombincleavage sites and factor Xa cleavage sites. TABLE 4 Conservative aminoacid substitutions Basic: arginine lysine histidine Acidic: glutamicacid aspartic acid Polar: glutamine asparagine Hydrophobic: leucineisoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small:glycine alanine serine threonine methionine

[0133] The proteins of the present invention can also comprisenon-naturally occurring amino acid residues. Non-naturally occurringamino acids include, without limitation, trans-3-methylproline,2,4-methanoproline, cis-4-hydroxy-proline, trans-4-hydroxyproline,N-methyl-glycine, allo-threonine, methylthreonine,hydroxyethyl-cysteine, hydroxyethylhomocysteine, nitroglutamine,homo-glutamine, pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, 3,3-dimethyl-proline,tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, and 4-fluoro-phenylalanine. Several methods areknown in the art for incorporating non-naturally occurring amino acidresidues into proteins. For example, an in vitro system can be employedwherein nonsense mutations are suppressed using chemically aminoacylatedsuppressor tRNAs. Methods for synthesizing amino acids andaminoacylating tRNA are known in the art. Transcription and translationof plasmids containing nonsense mutations is carried out in a cell-freesystem comprising an E. coli S30 extract and commercially availableenzymes and other reagents. Proteins are purified by chromatography.See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991;Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA90:10145-9, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-8, 1996). Within a third method, E. coli cells are cultured inthe absence of a natural amino acid that is to be replaced (e.g.,phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine) . The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993).

[0134] A limited number of non-conservative amino acids, amino acidsthat are not encoded by the genetic code, non-naturally occurring aminoacids, and unnatural amino acids may be substituted for zsig39 aminoacid residues.

[0135] Essential amino acids in the polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al., Proc.Natl. Acad. Sci. USA 88:4498-502, 1991). In the latter technique, singlealanine mutations are introduced at every residue in the molecule, andthe resultant mutant molecules are tested for biological activity (e.g.,ability to modulate energy balance) as disclosed below to identify aminoacid residues that are critical to the activity of the molecule. Seealso, Hilton et al., J. Biol. Chem. 271:4699-708, 1996. Sites ofligand-receptor or other biological interaction can also be determinedby physical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., Science 255.306-12,1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al.,FEBS Lett. 309:59-64, 1992. The identities of essential amino acids canalso be inferred from analysis of homologies with related polypeptides.

[0136] Multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner etal., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Neret al., DNA 7:127, 1988).

[0137] Variants of the disclosed zsig39 DNA and polypeptide sequencescan be generated through DNA shuffling as disclosed by Stemmer, Nature370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated byin vitro homologous recombination by random fragmentation of a parentDNA followed by reassembly using PCR, resulting in randomly introducedpoint mutations. This technique can be modified by using a family ofparent DNAs, such as allelic variants or DNAs from different species, tointroduce additional variability into the process. Selection orscreening for the desired activity, followed by additional iterations ofmutagenesis and assay provides for rapid “evolution” of sequences byselecting for desirable mutations while simultaneously selecting againstdetrimental changes.

[0138] Mutagenesis methods as disclosed above can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode active polypeptides (e.g., ability to modulateenergy balance) can be recovered from the host cells and rapidlysequenced using modern equipment. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest, and can be applied to polypeptides of unknownstructure.

[0139] Using the methods discussed above, one of ordinary skill in theart can identify and/or prepare a variety of polypeptides that aresubstantially homologous to residues 19 to 243 of SEQ ID NO:2 or allelicvariants thereof and retain the energy balance modulating or otherproperties of the wild-type protein. Such polypeptides may includeadditional amino acids, such as additional collagen repeats of theGly-Xaa-Pro or Gly-Xaa-Xaa type. Such polypeptides may also includeadditional polypeptide segments as generally disclosed above.

[0140] The polypeptides of the present invention, including full-lengthproteins, fragments thereof and fusion proteins, can be produced ingenetically engineered host cells according to conventional techniques.Suitable host cells are those cell types that can be transformed ortransfected with exogenous DNA and grown in culture, and includebacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryoticcells, particularly cultured cells of multicellular organisms, arepreferred. Techniques for manipulating cloned DNA molecules andintroducing exogenous DNA into a variety of host cells are disclosed bySambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, andAusubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley and Sons, Inc., NY, 1987.

[0141] In general, a DNA sequence encoding a zsig39 polypeptide of thepresent invention is operably linked to other genetic elements requiredfor its expression, generally including a transcription promoter andterminator within an expression vector. The vector will also commonlycontain one or more selectable markers and one or more origins ofreplication, although those skilled in the art will recognize thatwithin certain systems selectable markers may be provided on separatevectors, and replication of the exogenous DNA may be provided byintegration into the host cell genome. Selection of promoters,terminators, selectable markers, vectors and other elements is a matterof routine design within the level of ordinary skill in the art. Manysuch elements are described in the literature and are available throughcommercial suppliers.

[0142] To direct a zsig39 polypeptide into the secretory pathway of ahost cell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of the zsig39 polypeptide, ormay be derived from another secreted protein (e.g., t-PA) or synthesizedde novo. The secretory signal sequence is joined to the zsig39polypeptide DNA sequence in the correct reading frame and positioned todirect the newly synthesized polypeptide into he secretory pathway ofthe host cell. Secretory signal sequences are commonly positioned 5′ tothe DNA sequence encoding the polypeptide of interest, although certainsignal sequences may be positioned elsewhere in the DNA sequence ofinterest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland etal., U.S. Pat. No. 5,143,830).

[0143] Alternatively, the secretory signal sequence contained in thepolypeptides of the present invention is used to direct otherpolypeptides into the secretory pathway. The present invention providesfor such fusion polypeptides. A signal fusion polypeptide can be madewherein a secretory signal sequence derived from amino acid residues1-15 or 1-19 of SEQ ID NO:2 is be operably linked to another polypeptideusing methods known in the art and disclosed herein. The secretorysignal sequence contained in the fusion polypeptides of the presentinvention is preferably fused amino-terminally to an additional peptideto direct the additional peptide into the secretory pathway. Suchconstructs have numerous applications known in the art. For example,these novel secretory signal sequence fusion constructs can direct thesecretion of an active component of a normally non-secreted protein,such as a receptor. Such fusions may be used in vivo or in vitro todirect peptides through the secretory pathway.

[0144] Cultured mammalian cells are also suitable hosts within thepresent invention. Methods for introducing exogenous DNA into mammalianhost cells include dalcium phosphate-mediated transfection (Wigler etal., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics7:603, 1981: Graham and Van der Eb, Virology 52:456, 1973),electroporation (Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextranmediated transfection (Ausubel et al., eds., Current Protocols inMolecular Biology, John Wiley and Sons, Inc., NY, 1987),liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993;Ciccarone et al., Focus 15:80, 1993), and viral vectors (Miller andRosman, BioTechniques 7:980-90, 1989; Wang and Finer, Nature Med.2:714-16, 1996). The production of recombinant polypeptides in culturedmammalian cells is disclosed, for example, by Levinson et al., U.S. Pat.No. 4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al.,U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Preferredcultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7(ATCC No. CRL 1651), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection, Rockville, Md. In general,strong transcription promoters are preferred, such as promoters fromSV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

[0145] Drug selection is generally used to select for cultured mammaliancells into which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the ene of interestto their progeny are referred to as “stable transfectants.” A preferredselectable marker is gene encoding resistance to the antibioticneomycin. election is carried out in the presence of a neomycin-typedrug, such as G-418 or the like. Selection systems may also be used toincrease the expression level of the gene of interest, a processreferred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g., hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

[0146] Other higher eukaryotic cells can also be used as hosts,including plant cells, insect cells and avian cells. The use ofAgrobacterium rhizogenes as a vector for expressing genes in plant cellshas been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58,1987. Transformation of insect cells and production of foreignpolypeptides therein is disclosed by Guarino et al., U.S. Pat. No.5,162,222 and WIPO publication WO 94/06463. Insect cells can be infectedwith recombinant baculovirus, commonly derived from Autographacalifornica nuclear polyhedrosis virus (AcNPV). See, King and Possee,The Baculovirus Expression System: A Laboratory Guide, London, Chapman &Hall; O'Reilly et al., Baculovirus Expression Vectors: A LaboratoryManual, New York, Oxford University Press., 1994; and, Richardson, C.D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology,Totowa, N.J., Humana Press, 1995. A second method of making recombinantzsig39 baculovirus utilizes a transposon-based system described byLuckow (Luckow et al., J. Virol. 67:4566-79, 1993). This system, whichutilizes transfer vectors, is sold in the Bac-to-Bac™ kit (LifeTechnologies, Rockville, Md.). This system utilizes a transfer vector,pFastBacl™ (Life Technologies) containing a Tn7 transposon to move theDNA encoding the zsig39 polypeptide into a baculovirus genome maintainedin E. coli as a large plasmid called a “bacmid.” See, Hill-Perkins andPossee, J. Gen. Virol. 71:971-6, 1990; Bonning et al:, J. Gen. Virol.75:1551-6, 1994; and, Chazenbalk and Rapoport, J. Biol. Chem.270:1543-9, 1995. In addition, transfer vectors can include an in-framefusion with DNA encoding an epitope tag at the C- or N-terminus of theexpressed zsig39 polypeptide, for example, a Glu-Glu epitope tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985). Using atechnique known in the art, a transfer vector containing zsig39 istransformed into E. coli, and screened for bacmids which contain aninterrupted lacZ gene indicative of recombinant baculovirus. The bacmidDNA containing the recombinant baculovirus genome is isolated, usingcommon techniques, and used to transfect Spodoptera frugiperda cells,e.g. Sf9 cells. Recombinant virus that expresses zsig39 is subsequentlyproduced. Recombinant viral stocks are made by methods commonly used theart.

[0147] The recombinant virus is used to infect host cells, typically acell line derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commerciallyavailable serum-free media are used to grow and maintain the cells.Suitable media are Sf900 II™ (Life Technologies) or ESF 921™ (ExpressionSystems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa,Kans.) or Express FiveO™ (Life Technologies) for the T. ni cells. Thecells are grown up from an inoculation density of approximately 2-5×10⁵cells to a density of 1-2×10⁶ cells at which time a recombinant viralstock is added at a multiplicity of infection (MOI) of 0.1 to 10, moretypically near 3. Procedures used are generally described in availablelaboratory manuals (King and Possee, ibid.; O'Reilly et al., ibid.;Richardson, ibid.). Subsequent purification of the zsig39 polypeptidefrom the supernatant can be achieved using methods described herein.

[0148] Fungal cells, including yeast cells, can also be used within thepresent invention. Yeast species of particular interest in this regardinclude Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Methods for transforming S. cerevisiae cells with exogenousDNA and producing recombinant polypeptides therefrom are disclosed by,for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S.Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S.Pat. No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075.Transformed cells are selected by phenotype determined by the selectablemarker, commonly drug resistance or the ability to grow in the absenceof a particular nutrient (e.g., leucine). A preferred vector system foruse in Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349. Methods for transforming Acremoniumchrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228.Methods for transforming Neurospora are disclosed by Lambowitz, U.S.Pat. No. 4,486,533.

[0149] The use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed in WIPO Publications WO 97/17450, WO97/17451, WO 98/02536, and Wo 98/02565. DNA molecules for use intransforming P. methanolica will commonly be prepared asdouble-stranded, circular plasmids, which are preferably linearizedprior to transformation. For polypeptide production in P. methanolica,it is preferred that the promoter and terminator in the plasmid be thatof a P. methanolica gene, such as a P. methanolica alcohol utilizationgene (AUG1 or AUG2). Other useful promoters include those of thedihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), andcatalase (CAT) genes. To facilitate integration of the DNA into the hostchromosome, it is preferred to have the entire expression segment of theplasmid flanked at both ends by host DNA sequences. A preferredselectable marker for use in Pichia methanolica is a P. methanolica ADE2gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC;EC 4.1.1.21), which allows ade2 host cells to grow in the absence ofadenine. For large-scale, industrial processes where it is desirable tominimize the use of methanol, it is preferred to use host cells in whichboth methanol utilization genes (AUG1 and AUG2) are deleted. Forproduction of secreted proteins, host cells deficient in vacuolarprotease genes (PEP4 and PRB1) are preferred. Electroporation is used tofacilitate the introduction of a plasmid containing DNA encoding apolypeptide of interest into P. methanolica cells. It is preferred totransform P. Pmethanolica cells by electroporation using anexponentially decaying, pulsed electric field having a field strength offrom 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant(τ) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.

[0150] Prokaryotic host cells, including strains of the bacteriaEscherichia coli, Bacillus and other genera are also useful host cellswithin the present invention. Techniques for transforming these hostsand expressing foreign DNA sequences cloned therein are well known inthe art (see, e.g., Sambrook et al., ibid.). When expressing a zsig39polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

[0151] Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell.

[0152] Expressed recombinant zsig39 polypeptides (or chimeric zsig39polypeptides) can be purified using fractionation and/or conventionalpurification methods and media. Ammonium sulfate precipitation and acidor chaotrope extraction may be used for fractionation of samples.Exemplary purification steps may include hydroxyapatite, size exclusion,FPLC and reverse-phase high performance liquid chromatography. Suitableanion exchange media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are preferred, with DEAE Fast-Flow Sepharose (Pharmacia,Piscataway, N.J.) being particularly preferred. Suitable chromatographicmedia include those media derivatized with phenyl, butyl, or octylgroups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650(Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and thelike; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) andthe like. Suitable solid supports include glass beads, silica-basedresins, cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like thatare insoluble under the conditions in which they are to be used. Thesesupports may be modified with reactive groups that allow attachment ofproteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxylgroups and/or carbohydrate moieties. Examples of coupling chemistriesinclude cyanogen bromide activation, N-hydroxysuccinimide activation,epoxide activation, sulfhydryl activation, hydrazide activation, andcarboxyl and amino derivatives for carbodiimide coupling chemistries.These and other solid media are well known and widely used in the art,and are available from commercial suppliers. Methods for bindingreceptor polypeptides to support media are well known in the art.Selection of a particular method is a matter of routine design and isdetermined in part by the properties of the chosen support. See, forexample, Affinity Chromatography: Principles & Methods, Pharmacia LKBBiotechnology, Uppsala, Sweden, 1988.

[0153] The polypeptides of the present invention can be isolated byexploitation of their structural or binding properties. For example,immobilized metal ion adsorption (IMAC) chromatography can be used topurify histidine-rich proteins, or proteins having His tags. Briefly, agel is first charged with divalent metal ions to form a chelate(Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-rich proteinswill be adsorbed to this matrix with differing affinities, dependingupon the metal ion used, and will be eluted by competitive elution,lowering the pH, or use of strong chelating agents. Other methods ofpurification include purification of glycosylated proteins by lectinaffinity chromatography and ion exchange chromatography (Methods inEnzymol., Vol. 182, “Guide to Protein Purification”, M. Deutscher,(ed.), Acad. Press, San Diego, 1990, pp.529-39). Within additionalembodiments of the invention, a fusion of the polypeptide of interestand an affinity tag (e.g., Glu-Glu affinity tags, FLAG tags,maltose-binding protein, an immunoglobulin domain) may be constructed tofacilitate purification. Such purification methods are disclosed indetain in the Example section below.

[0154] Protein refolding (and optionally reoxidation) procedures may beadvantageously used. It is preferred to purify the protein to >80%purity, more preferably to >90% purity, even more preferably >95%, andparticularly preferred is a pharmaceutically pure state, that is greaterthan 99.9w pure with respect to contaminating macromolecules,particularly other proteins and nucleic acids, and free of infectiousand pyrogenic agents. Preferably, a purified protein is substantiallyfree of other proteins, particularly other proteins of animal origin.

[0155] Zsig39 polypeptides or fragments thereof may also be preparedthrough chemical synthesis. Such zsig39 polypeptides may be monomers ormultimers; glycosylated or non-glycosylated; pegylated or non-pegylated;and may or may not include an initial methionine amino acid residue.

[0156] An in vivo approach for assaying proteins of the presentinvention involves viral delivery systems. Exemplary viruses for thispurpose include adenovirus, herpesvirus, vaccinia virus andadeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus,is currently the best studied gene transfer vector for delivery ofheterologous nucleic acid (for a review, see Becker et al., Meth. CellBiol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine4:44-53, 1997). The adenovirus system offers several advantages:adenovirus can (i) accommodate relatively large DNA inserts; (ii) begrown to high-titer; (iii) infect a broad range of mammalian cell types;and (iv) be used with a large number of available vectors containingdifferent promoters. Also, because adenoviruses are stable in thebloodstream, they can be administered by intravenous injection.

[0157] By deleting portions of the adenovirus genome, larger inserts (upto 7 kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. In an exemplary system, theessential El gene has been deleted from the viral vector, and the viruswill not replicate unless the E1 gene is provided by the host cell (thehuman 293 cell line is exemplary). When intravenously administered tointact animals, adenovirus primarily targets the liver. If theadenoviral delivery system has an E1 gene deletion, the virus cannotreplicate in the host cells. However, the host's tissue (e.g., liver)will express and process (and, if a secretory signal sequence ispresent, secrete) the heterologous protein. Secreted proteins will enterthe circulation in the highly vascularized liver, and effects on theinfected animal can be determined.

[0158] The adenovirus system can also be used for protein production invitro. By culturing adenovirus-infected non-293 cells under conditionswhere the cells are not rapidly dividing, the cells can produce proteinsfor extended periods of time. For instance, BHK cells are grown toconfluence in cell factories, then exposed to the adenoviral vectorencoding the secreted protein of interest. The cells are then grownunder serum-free conditions, which allows infected cells to survive forseveral weeks without significant cell division. Alternatively,adenovirus vector infected 293S cells can be grown in suspension cultureat relatively high cell density to produce significant amounts ofprotein (see Garnier et al., Cytotechnol. 15:145-55, 1994). With eitherprotocol, an expressed, secreted heterologous protein can be repeatedlyisolated from the cell culture supernatant. Within the infected 293Scell production protocol, non-secreted proteins may also be effectivelyobtained.

[0159] A ligand-binding polypeptide, such as a zsig39polypeptide-binding polypeptide, can also be used for purification ofligand. The polypeptide is immobilized on a solid support, such as beadsof agarose, cross-linked agarose, glass, cellulosic resins, silica-basedresins, polystyrene, cross-linked polyacrylamide, or like materials thatare stable under the conditions of use. Methods for linking polypeptidesto solid supports are known in the art, and include amine chemistry,cyanogen bromide activation, N-hydroxysuccinimide activation, epoxideactivation, sulfhydryl activation, and hydrazide activation. Theresulting medium will generally be configured in the form of a column,and fluids containing ligand are passed through the column one or moretimes to allow ligand to bind to the ligand-binding polypeptide. Theligand is then eluted using changes in salt concentration, chaotropicagents (guanidine HCl), or pH to disrupt ligand-receptor binding.

[0160] An assay system that uses a ligand-binding receptor (or anantibody, one member of a complement/anti-complement pair) or a bindingfragment thereof, and a commercially available biosensor instrument(BIAcore™, Pharmacia Biosensor, Piscataway, N.J.) may be advantageouslyemployed. Such receptor, antibody, member of acomplement/anti-complement pair or fragment is immobilized onto thesurface of a receptor chip. Use of this instrument is disclosed byKarlsson, J. Immunol. Methods 145:229-40, 1991 and Cunningham and Wells,J. Mol. Biol. 234:554-63, 1993. A receptor, antibody, member or fragmentis covalently attached, using amine or sulfhydryl chemistry, to dextranfibers that are attached to gold film within the flow cell. A testsample is passed through the cell. If a ligand, epitope, or oppositemember of the complement/anti-complement pair is present in the sample,it will bind to the immobilized receptor, antibody or member,respectively, causing a change in the refractive index of the medium,which is detected as a change in surface plasmon resonance of the goldfilm. This system allows the determination of on- and off-rates, fromwhich binding affinity can be calculated, and assessment ofstoichiometry of binding.

[0161] Ligand-binding polypeptides can also be used within other assaysystems known in the art. Such systems include Scatchard analysis fordetermination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51:660-72, 1949) and calorimetric assays (Cunningham et al., Science253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).

[0162] As would be evident to one of ordinary skill in the art,polyclonal antibodies can be generated from inoculating a variety ofwarm-blooded animals such as horses, cows, goats, sheep, dogs, chickens,rabbits, mice, hamsters, guinea pigs and rats as well as transgenicanimals such as transgenic sheep, cows, goats or pigs. Antibodies mayalso be expressed in yeast and fungi in modified forms as well as inmammalian and insect cells. The zsig39 polypeptide or a fragment thereofserves as an antigen (immunogen) to inoculate an animal or elicit animmune response. Suitable antigens would include the zsig39 polypeptideencoded by SEQ. ID NO:2 from amino acid residue 16-2243 of SEQ ID NO:2,from amino acid residue 19-243 of SEQ ID NO:2, or a contiguous 9-243amino acid residue fragment thereof. The immunogenicity of a zsig39polypeptide may be increased through the use of an adjuvant, such asalum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.Polypeptides useful for immunization also include fusion polypeptides,such as fusions of zsig39 or a portion thereof with an immunoglobulinpolypeptide or with an affinity tag. The polypeptide immunogen may be afull-length molecule or a portion thereof. If the polypeptide portion is“hapten-like”, such portion may be advantageously joined or linked to amacromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovineserum albumin (BSA) or tetanus toxoid) for immunization.

[0163] As used herein, the term “antibodies” includes polyclonalantibodies, affinity-purified polyclonal antibodies, monoclonalantibodies, and antigen-binding fragments, such as F(ab′)₂ and Fabproteolytic fragments. Genetically engineered intact antibodies orfragments, such as chimeric antibodies, Fv fragments, single chainantibodies and the like, as well as synthetic antigen-binding peptidesand polypeptides, are also included. Non-human antibodies may behumanized by grafting only non-human CDRs onto human framework andconstant regions, or by incorporating the entire non-human variabledomains (optionally “cloaking” them with a human-like surface byreplacement of exposed residues, wherein the result is a “veneered”antibody). In some instances, humanized antibodies may retain non-humanresidues within the human variable region framework domains to enhanceproper binding characteristics. Through humanizing antibodies,biological half-life may be increased, and the potential for adverseimmune reactions upon administration to humans is reduced. Alternativetechniques for generating or selecting antibodies useful herein includein vitro exposure of lymphocytes to zsig39 protein or peptide, andselection of antibody display libraries in phage or similar vectors (forinstance, through use of immobilized or labeled zsig39 protein orpeptide).

[0164] Antibodies are defined to be specifically binding if: 1) theyexhibit a threshold level of binding activity, and/or 2) they do notsignificantly cross-react with related polypeptide molecules. First,antibodies herein specifically bind if they bind to a zsig39polypeptide, peptide or epitope with a binding affinity (K_(a)) of 10⁶mol⁻¹ or greater, preferably 10⁷ mol⁻¹ or greater, more preferably 10⁸mol⁻¹ or greater, and most preferably 10⁹ mol⁻¹ or greater. The bindingaffinity of an antibody can be readily determined by one of ordinaryskill in the art, for example, by Scatchard analysis (Scatchard, Ann. NYAcad. Sci. 51: 660-72, 1949).

[0165] Second, antibodies specifically bind if they do not significantlycross-react with related polypeptides. Antibodies do not significantlycross-react with related polypeptide molecules, for example, if theydetect zsig39 polypeptide but not known related polypeptides using astandard Western blot analysis (Ausubel et al., ibid.). Examples ofknown related polypeptides are orthologs, proteins from the same speciesthat are members of a protein family such as Acrp30 (SEQ ID NO:8), thepolypeptides shown in alignment FIG. 1, mutant human zsig39polypeptides, and the like. Moreover, antibodies may be “screenedagainst” known related polypeptides to isolate a population thatspecifically binds to the inventive polypeptides. For example,antibodies raised to human zsig39 polypeptides are adsorbed to relatedpolypeptides adhered to insoluble matrix; antibodies specific to humanzsig39 polypeptides will flow through the matrix under the proper bufferconditions. Such screening allows isolation of polyclonal and monoclonalantibodies non-crossreactive to closely related polypeptides(Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988; Current Protocols in Immunology,Cooligan, et al. (eds.), National Institutes of Health, John Wiley andSons, Inc., 1995). Screening and isolation of specific antibodies iswell known in the art (see, Fundamental Immunology, Paul (eds.), RavenPress, 1993; Getzoff et al., Adv. in Immunol. 43: 1-98, 1988; MonoclonalAntibodies: Principles and Practice, Goding, J. W. (eds.), AcademicPress Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101, 1984).Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmuno-assay, radioimmuno-precipitation,enzyme-linked immuno-sorbent assay (ELISA), dot blot or Western blotassay, inhibition or competition assay, and sandwich assay.

[0166] Genes encoding polypeptides having potential zsig39 polypeptidebinding domains, “binding proteins”, can be obtained by screening randomor directed peptide libraries displayed on phage (phage display) or onbacteria, such as E. coli. Nucleotide sequences encoding thepolypeptides can be obtained in a number of ways, such as through randommutagenesis and random polynucleotide synthesis. Alternatively,constrained phage display libraries can also be produced. These peptidedisplay libraries can be used to screen for peptides which interact witha known target which can be a protein or polypeptide, such as a ligandor receptor, a biological or synthetic macromolecule, or organic orinorganic substances. Techniques for creating and screening such peptidedisplay libraries are known in the art (Ladner et al., U.S. Pat. No.5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S.Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) andpeptide display libraries and kits for screening such libraries areavailable commercially, for instance from Clontech (Palo Alto, Calif.),Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly,Mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.). Peptidedisplay libraries can be screened using the zsig39 sequences disclosedherein to identify proteins which bind to zsig39. These “bindingproteins” which interact with zsig39 polypeptides can be usedessentially like an antibody, for tagging cells; for isolating homologpolypeptides by affinity purification; directly or indirectly conjugatedto drugs, toxins, radionuclides and the like. These binding proteins canalso be used in analytical methods such as for screening expressionlibraries and neutralizing activity. The binding proteins can also beused for diagnostic assays for determining circulating levels ofpolypeptides; for detecting or quantitating soluble polypeptides asmarker of underlying pathology or disease. To increase the half-life ofthese binding proteins, they can be conjugated. Their biologicalproperties may be modified by dimerizing or multimerizing for use asagonists or antagonists. Binding peptides can be screened against knownrelated polypeptides as described above.

[0167] Antibodies and binding proteins to zsig39 may be used for taggingcells that express zsig39; for isolating zsig39 by-affinitypurification; for diagnostic assays for determining circulating levelsof zsig39 polypeptides; for detecting or quantitating soluble zsig39 asmarker of underlying pathology or disease; in analytical methodsemploying FACS; for screening expression libraries; for generatinganti-idiotypic antibodies; and as neutralizing antibodies or asantagonists to block zsig39 polypeptide energy balance modulationactivity or like activity in vitro and in vivo. Suitable direct tags orlabels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent markers, chemiluminescent markers, magneticparticles and the like; indirect tags or labels may feature use ofbiotin-avidin or other complement/anti-complement pairs asintermediates. Moreover, antibodies to zsig39 or fragments thereof maybe used in vitro to detect denatured zsig39 or fragments thereof inassays, for example, Western Blots or other assays known in the art.

[0168] Antibodies or binding proteins herein can also be directly orindirectly conjugated to drugs, toxins, radionuclides and the like, andthese conjugates used for in vivo diagnostic or therapeuticapplications. For instance, polypeptides or antibodies of the presentinvention can be used to identify or treat tissues or organs thatexpress a corresponding anti-complementary molecule (receptor orantigen, respectively, for instance). More specifically, zsig39polypeptides or anti-zsig39 antibodies, or bioactive fragments orportions thereof, can be coupled to detectable or cytotoxic moleculesand delivered to a mammal having cells, tissues or organs that expressthe anti-complementary molecule.

[0169] Suitable detectable molecules may be directly or indirectlyattached to the polypeptide or antibody, and include radionuclides,enzymes, substrates, cofactors, inhibitors, fluorescent markers,chemiluminescent markers, magnetic particles and the like. Suitablecytotoxic molecules may be directly or indirectly attached to thepolypeptide or antibody, and include bacterial or plant toxins (forinstance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and thelike), as well as therapeutic radionuclides, such as iodine-131,rhenium-188 or yttrium-90 (either directly attached to the polypeptideor antibody, or indirectly attached through means of a chelating moiety,for instance). Polypeptides or antibodies may also be conjugated tocytotoxic drugs, such as adriamycin. For indirect attachment of adetectable or cytotoxic molecule, the detectable or cytotoxic moleculecan be conjugated with. a member of a complementary/anti-complementarypair, where the other member is bound to the polypeptide or antibodyportion. For these purposes, biotin/streptavidin is an exemplarycomplementary/anticomplementary pair.

[0170] In another embodiment, polypeptide-toxin fusion proteins orantibody-toxin fusion proteins can be used for targeted cell or tissueinhibition or ablation (for instance, to treat cancer cells or tissues).Alternatively, if the polypeptide has multiple functional domains (i.e.,an activation domain or a ligand binding domain, plus a targetingdomain), a fusion protein including only the targeting domain may besuitable for directing a detectable molecule, a cytotoxic molecule or acomplementary molecule to a cell or tissue type of interest. Ininstances where the domain only fusion protein includes a complementarymolecule, the anti-complementary molecule can be conjugated to adetectable or cytotoxic molecule. Such domain-complementary moleculefusion proteins thus represent a generic targeting vehicle forcell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates. Thebioactive polypeptide or antibody conjugates described herein can bedelivered intravenously, intraarterially, intraductally with DMSO,intramuscularly, subcutaneously, intraperitoneally, also by transdermalmethods, by electro-transfer, orally or via inhalant.

[0171] Polynucleotides encoding zsig39 polypeptides are useful withingene therapy applications where it is desired to increase or inhibitzsig39 activity. If a mammal has a mutated or absent zsig39 gene, thezsig39 gene can be introduced into the cells of the mammal. In oneembodiment, a gene encoding a zsig39 polypeptide is introduced in vivoin a viral vector. Such vectors include an attenuated or defective DNAvirus, such as, but not limited to, herpes simplex virus (HSV),papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associatedvirus (AAV), and the like. Defective viruses, which entirely or almostentirely lack viral genes, are preferred. A defective virus is notinfective after introduction into a cell. Use of defective viral vectorsallows for administration to cells in a specific, localized area,without concern that the vector can infect other cells. Examples ofparticular vectors include, but are not limited to, a defective herpessimplex virus 1 (HSVl) vector (Kaplitt et al., Molec. Cell. Neurosci.2:320-30, 1991); an attenuated adenovirus vector, such as the vectordescribed by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-30,1992; and a defective adeno-associated virus vector (Samulski et al., J.Virol. 61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).

[0172] In another embodiment, a zsig39 gene can be introduced in aretroviral vector, e.g., as described in Anderson et al., U.S. Pat. No.5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Pat. No.4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J.Virol. 62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263; WIPOPublication WO 95/07358; and Kuo et al., Blood 82:845, 1993.Alternatively, the vector can be introduced by lipofection in vivo usingliposomes. Synthetic cationic lipids can be used to prepare liposomesfor in vivo transfection of a gene encoding a marker (Felgner et al.,Proc. Natl. Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl.Acad. Sci. USA 85:8027-31, 1988). The use of lipofection to introduceexogenous genes into specific organs in vivo has certain practicaladvantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. More particularly, directingtransfection to particular cells represents one area of benefit. Forinstance, directing transfection to particular cell types would beparticularly advantageous in a tissue with cellular heterogeneity, suchas the pancreas, liver, kidney, and brain. Lipids may be chemicallycoupled to other molecules for the purpose of targeting. Targetedpeptides (e.g., hormones or neurotransmitters), proteins such asantibodies, or non-peptide molecules can be coupled to liposomeschemically.

[0173] It is possible to remove the target cells from the body; tointroduce the vector as a naked DNA plasmid; and then to re-implant thetransformed cells into the body. Naked DNA vectors for gene therapy canbe introduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem.267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.

[0174] Antisense methodology can be used to inhibit zsig39 genetranscription, such as to inhibit cell proliferation in vivo.Polynucleotides that are complementary to a segment of a zsig39-encodingpolynucleotide (e.g., a polynucleotide as set froth in SEQ ID NO:1) aredesigned to bind to zsig39-encoding mRNA and to inhibit translation ofsuch mRNA. Such antisense polynucleotides are used to inhibit expressionof zsig39 polypeptide-encoding genes in cell culture or in a subject.

[0175] Transgenic mice, engineered to express the zsig39 gene, and micethat exhibit a complete absence of zsig39 gene function, referred to as“knockout mice” (Snouwaert et al., Science 257:1083, 1992), may also begenerated (Lowell et al., Nature 366:740-42, 1993). These mice may beemployed to study the zsig39 gene and the protein encoded thereby in anin vivo system.

[0176] For pharmaceutical use, the proteins of the present invention areformulated for parenteral, particularly intravenous or subcutaneous,delivery according to conventional methods. Intravenous administrationwill be by bolus injection or infusion over a typical period of one toseveral hours. In general, pharmaceutical formulations will include azsig39 protein in combination with a pharmaceutically acceptablevehicle, such as saline, buffered saline, .5% dextrose in water or thelike. Formulations may further include one or more excipients,preservatives, solubilizers, buffering agents, albumin to preventprotein loss on vial surfaces, etc. Methods of formulation are wellknown in the art and are disclosed, for example, in Remington: TheScience and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,Easton Pa., 19^(th) ed., 1995. Therapeutic doses will generally bedetermined by the clinician according to accepted standards, taking intoaccount the nature and severity of the condition to be treated, patienttraits, etc. Determination of dose is within the level of ordinary skillin the art.

[0177] The invention is further illustrated by the followingnon-limiting examples.

EXAMPLE 1 Extension of EST Sequence

[0178] The novel zsig39 polypeptide-encoding polynucleotides of thepresent invention were initially identified by selecting an EST from anEST database, predicting a protein sequence based thereupon, andsearching known sequence databases for the secreted protein that is mosthomologous to predicted protein based on the EST. ESTs that potentiallyencode proteins having biologically interesting homology to knownsecreted proteins were identified for further study. A single ESTsequence was discovered and predicted to be homologous to adipocytespecific protein. See, for example, Scherer et al., J. Biol. Chem.270(45): 26746-9, 1995. To identify the corresponding cDNA, a cloneconsidered likely to contain the entire coding sequence was used forsequencing. Using an Invitrogen S.N.A.P.™ Miniprep kit (Invitrogen,Corp., San Diego, Calif.) according to manufacturer's instructions a 5ml overnight culture in LB+50 μg/ml ampicillin was prepared. Thetemplate was sequenced on an ABIPRISM ™ model 377 DNA sequencer(Perkin-Elmer Cetus, Norwalk, Conn.) using the ABI PRISM™ Dye TerminatorCycle Sequencing Ready Reaction Kit (Perkin-Elmer Corp.) according tomanufacturer's instructions. Oligonucleotides ZC447 (SEQ ID NO:11),ZC976 (SEQ ID NO:12) to the M13 and lacZ promoters on theclone-containing vector were used as sequencing primers.Oligonucleotides ZC14707 (SEQ ID NO:13), ZC14708 (SEQ ID NO:14), ZC14760(SEQ ID NO:15), ZC14758 (SEQ ID NO:16) and ZC14759 (SEQ ID NO:17) wereused to complete the sequence from the clone. Sequencing reactions werecarried out in a Hybaid OmniGene Temperature Cycling System (NationalLabnet Co., Woodbridge, N.Y.). SEQUENCHER™ 3.1 sequence analysissoftware (Gene Codes Corporation, Ann Arbor, Mich.) was used for dataanalysis. The resulting 1347 bp sequence is disclosed in SEQ ID NO: 1.Comparison of the originally derived EST sequence with the sequencerepresented in SEQ ID NO:1 showed that there were 27 base pairdifferences which resulted in 11 amino acid differences between thededuced amino acid sequences. Note that 22 of the base pair differenceswere from unknown “N” residues in the EST sequence to known residues inSEQ ID NO:1, which result in “assumed” amino acid changes.

EXAMPLE 2 Tissue Distribution

[0179] Northerns were performed using Human Multiple Tissue Blots fromClontech (Palo Alto, Calif.). An approximately 1347 bp DNA probe,corresponding to the a sequence encompassing a polynucleotide encodingfull length zsig39 polypeptide, generated by EcoRl-NotI digest of theplasmid DNA. The resulting fragment was gel purified for use as a probe.The DNA probe was radioactively labeled with ³² P using REDIPRIME® DNAlabeling system (Amersham, Arlington Heights, Ill.) according to themanufacturer's specifications. The probe was purified using a NUCTRAPpush column (Stratagene Cloning Systems, La Jolla, Calif.). EXPRESSHYB(Clontech, Palo Alto, Calif.) solution was used for prehybridization andas a hybridizing solution for the Northern blots. Hybridization tookplace overnight at 65° C, and the blots were then washed in 2×SSC and0.1% SDS at room temperature, followed by a wash in 0.1×SSC and 0.1% SDSat 65° C. One transcript size was observed at approximately 1.2 kb.Signal intensity was highest for small intestine and heart, withrelatively less intense signals in pancreas, skeletal muscle, kidney andthyroid, and with lower intensity signals in placenta, lung, liver,spleen, prostate, ovary, colon, stomach, spinal cord, lymph node,trachea, adrenal gland and bone marrow.

[0180] Additional Northern Blot Analysis was done using a Cut NorthernTissue Blot. The blot was prepared using mRNA from human colorectaladenocarcinoma cell line SW480 (Clontech, Palo Alto, Calif.), humansmall intestine tissue (Clontech), human stomach tissue (Clontech),human intestinal smooth muscle cell line (Hism; ATCC No. CRL-1692;American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.),normal human colon cell line (FHC; ATCC No. CRL-1831; American TypeCulture Collection) and human normal fetal small intestine cell line(FHs74 Int.; ATCC No. CCL241; American Type Culture Collection).

[0181] Total RNAs were isolated from Hism, FHC and FHs74 int. by acidguanidium method (Cheomczynski et al., Anal. Biochem. 162:156-9, 1987).The polyA RNAs were selected by eluting total RNA through a column thatretains polyA⁺ RNAs (Aviv et al., Proc. Nat. Acad. Sci. 69:1408-12,1972). 2 μg of polyA⁺ RNA from each sample was separated out in a 1.5%agarose gel in 2.2 M formaldehyde and phosphate buffer. The RNAs weretransferred onto Nytran membrane (Schleicher and Schuell, Keene, N.H.)in 20×SSC overnight. The blot was treated in the UV Stratalinker 2400(Stratagene, La Jolla, Calif.) at 0.12 Joules. The bolt was then bakedat 80° C. for one hour.

[0182] The Northern blots were probed with the zsig39 PCR fragment(described below in Example 4) encoding the mature zsig39 polypeptide,which was radiolabeled with ³²p dCTP using a Rediprime pellet kit(Amersham, Arlington Heights, Ill.) according to the manufacturer'sspecifications. The blot was hybridized in EXPRESSHYB (Clontech) at 56°C. overnight. The blot was washed at room temperature in 2×SSC and 0.1%SDS, then in 2×SSC and 0.1% SDS at 65° C., and finally at 65° C. in0.1×SSC and 0.1% SDS. Results showed that zsig39 hybridized to alltissues except the human intestinal smooth muscle cell line HISM.

EXAMPLE 3 Chromosomal Mapping of the Zsig39 Gene

[0183] The zsig39 polypeptide-encoding gene was mapped to chromosome 11using the commercially available “GeneBridge 4 Radiation Hybrid Panel”(Research Genetics, Inc., Huntsville, Ala.). The GeneBridge 4 RadiationHybrid Panel contains PCRable DNAs from each of 93 radiation hybridclones, plus two control DNAs (the HFL donor and the A23 recipient). Apublicly available WWW server(http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) allows mappingrelative to the Whitehead Institute/MIT Center for Genome Research'sradiation hybrid map of the human genome (the “WICGR” radiation hybridmap) which was constructed with the GeneBridge 4 Radiation Hybrid Panel.

[0184] For the mapping of the zsig39 gene with the “GeneBridge 4 RHPanel”, 20 μl reactions were set up in a PCRable 96-well microtiterplate (Stratagene, La Jolla, Calif.) and used in a “RoboCycler Gradient96” thermal cycler (Stratagene). Each of the 95 PCR reactions consistedof 2 μl 10× KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc.,Palo Alto, Calif.), 1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, FosterCity, Calif.), 1 μl sense primer, ZC15002 (SEQ ID NO:18), 1 μl antisenseprimer, ZC15003 (SEQ ID NO:19), 2 μl RediLoad (Research Genetics, Inc.,Huntsville, Ala.), 0.4 μl 50× Advantage KlenTaq Polymerase Mix (ClontechLaboratories, Inc.), 25 ng of DNA from an individual hybrid clone orcontrol and ddH₂O for a total volume of 20 μl. The reactions wereoverlaid with an equal amount of mineral oil and sealed. The PCR cyclerconditions were as follows: an initial 1 cycle 5 minute denaturation at95° C., 40 cycles of a 1 minute denaturation at 95° C., 1 minuteannealing at 64° C. and 1.5 minute extension at 72° C., followed by afinal 1 cycle extension of 7 minutes at 72° C. The reactions wereseparated by electrophoresis on a 2% agarose gel (Life Technologies,Gaithersburg, Md.).

[0185] The results showed that the zsig39 polypeptide-encoding gene maps549.99 cR_(—)3000 from the top of the human chromosome 11 linkage groupon the WICGR radiation hybrid map. Proximal and distal framework markerswere AFMB048ZA9 and FB17D4, respectively. The use of surrounding markerspositions the zsig39 gene in the 11q23.3 region on the integrated LDBchromosome 11 map (The Genetic Location Database, University ofSouthhampton, WWW server: http://cedar.genetics.soton.ac.uk/public_html/).

EXAMPLE 4 Construction of zsig39 Mammalian Expression Vectorszsig39CEE/pZP9 and zsig39NEE/pZP9

[0186] Two expression vectors were prepared for the zsig39 polypeptide,zsig39CEE/pZP9 and zsig39NEE/pZP9, wherein the constructs are designedto express a zsig39 polypeptide with a C- or N-terminal Glu-Glu tag (SEQID NO:20).

[0187] Zsig39NEE/pZP9

[0188] A 690 bp PCR generated zsig39 DNA fragment was created usingZC15037 (SEQ ID NO:21) and ZC15038 (SEQ ID NG:22) as PCR primers andcolonies described above as a template. An N-terminal Glu-Glu tag andrestriction sites Bam HI and Xba I are added. PCR amplification of thezsig39 fragment were 94° C. for 90 seconds, 5 cycles of 94° C. for 10seconds, 34° C. for 20 seconds, 74° C. for 40 seconds followed by 25cycles at 94° C. for 10 seconds, 68° C. for 20 seconds, 72° C. for 40seconds, followed by a 5 minute extension at 72° C. A band of thepredicted size, 690 bp, was visualized by 1% agarose gelelectrophoresis, excised and the DNA was purified from the gel with aQUIAQUICK column (Qiagen) according the manufacturer's instructions. TheDNA was digested with the restriction enzymes Bam HI and Xba I, followedby extraction and precipitated.

[0189] The excised DNA was subcloned into plasmid pZP9 which had beencut with Bam HI and Xba I. The zsig39NEE/pZP9 expression vectorincorporates the TPA leader and the Glu-Glu epitope (SEQ ID NO:20) isattached at the N-terminus as a purification aid. Plasmid pZP9(deposited at the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md., ATCC No. 98668) is a mammalian expression vectorcontaining an expression cassette having the mouse metallothionein-1promoter, multiple restriction sites for insertion of coding sequences,a stop codon and a human growth hormone terminator. The plasmid also hasan E. coli origin of replication, a mammalian selectable markerexpression unit having an SV40 promoter, enhancer and origin ofreplication, a DHFR gene and the SV40 terminator.

[0190] 30 ng of the restriction digested N-terminal Glu-Glu-zsig39insert and 48 ng of the digested vector were ligated overnight at 16° C.One microliter of each ligation reaction was independentlyelectroporated into DH10B competent cells (GIBCO BRL, Gaithersburg, Md.)according to manufacturer's direction and plated onto LB platescontaining 50 mg/ml ampicillin, and incubated overnight. Colonies werescreened by PCR using primers ZC13006 (SEQ ID NO:23) and ZC13007 (SEQ IDNO:24). PCR screening was done at 94° C. for 4 minutes, 25 cycles of 94°C. for 30 seconds, 64° C. for 20 seconds, 72° C. for 1 minute, followedby a 10 minute extension at 72° C. Positive clones were plated on to LBAmp plates as above. The insert sequence of positive clones was verifiedby sequence analysis. A large scale plasmid preparation was done using aQIAGEN® Maxi prep kit (Qiagen) according to manufacturer's instructions.

[0191] Zsig39CEE/pZP9

[0192] A 744 bp PCR generated zsig39 DNA fragment was created inaccordance with the procedure set forth above using ZC15609 (SEQ IDNO:25) and ZC15232 (SEQ ID NO:26) as PCR primers to add the C-terminalGlu-Glu tag and Eco RI and Bam HI restriction sites. PCR amplificationwas done at 94° C. for 3 minutes, 5 cycles of 94° C. for 30 seconds, 30°C. for 20 seconds, 72° C. for 1 minute, 25 cycles at 94° C. for 30seconds, 64° C. for 20 seconds, 72° C. for 1 minute, followed by a 5minute extension at 72° C. The purified PCR fragment was digested withthe restriction enzymes Eco RI and Bamr HI, followed by extraction andprecipitation.

[0193] The excised zig39 DNA was subcloned into plasmid pZP9 which hadbeen cut with Eco RI and Bam HI. The zsig39CEE/pZP9 expression vectoruses the native zsig39 signal peptide and attaches the Glu-Glu tag (SEQID NO:20) to the C-terminal of the zsig39 polypeptide-encodingpolynucleotide sequence.

[0194] Thirty four ng of the restriction digested C-terminalGlu-Glu-zsig39 insert and 48 ng of the corresponding vector were ligatedinto DH10B cells and positive colonies were screened as described above.Positive clones were plated on to LB Amp plates as above. The insertsequence of positive clones were verified by sequence analysis. A largescale plasmid preparation was done using a QIAGEN® Maxi prep kit(Qiagen) according to manufacturer's instructions.

EXAMPLE 5 Transfection and Expression of zsig39NEE and CEE Polypeptides

[0195] BHK 570 cells (ATCC No. CRL-10314) were plated in 10 cm tissueculture dishes and allowed to grow to approximately 50 to 70% confluencyovernight at 37° C., 5% CO₂, in DMEM/FBS media (DMEM, Gibco/BRL HighGlucose, (Gibco BRL, Gaithersburg, Md.), 5%, fetal bovine serum(Hyclone, Logan, Utah), 2 μM L-glutamine (JRH Biosciences, Lenexa,Kans.), 1 μM sodium pyruvate (Gibco BRL)). The cells were thentransfected with the plasmid zsig39NEE/pZP9 (N-terminal Glu-Glu tag) orzsig39CEE/pZP9 (C-terminal Glu-Glu tag), using Lipofectamine™ (GibcoBRL), in serum free (SF) media formulation (DMEM, Gibco/BRL HighGlucose, (Gibco BRL, Gaithersburg, Md.), 2 mM L-glutamine, 2 mM sodiumpyruvate, 10 ug/ml transferrin, 5 μg/ml insulin, 10 μg/ml fetuin and 2ng/ml selenium). Sixteen micrograms of zsig39NEE/pZP9 and 16 tg ofzsig39CEE/pZP9 were separately diluted into 15 ml tubes to a total finalvolume of 640 μl SF media. In separate tubes, 35 μl of Lipofectamine™(Gibco BRL) was mixed with 605 μl of SF medium. The Lipofectamine™ mixwas added, to the DNA mix and allowed to incubate approximately 30minutes at room temperature. Five milliliters of SF media was added tothe DNA:Lipofectamine™ mixture. The cells were rinsed once with 5 ml ofSF media, aspirated, and the DNA:Lipofectamine™ mixture was added. Thecells were incubated at 37° C. for five hours, then 6.4 ml of DMEM/10%FBS, 1% PSN media was added to the plate. The plate was incubated at 37°C. overnight and the DNA:Lipofectamine™ mixture was replaced with freshFBS/DMEM media the next day. On day 2 post-transfection, the cells weresplit into the selection media (ESTEP #1 with 1 μM MTX) in 150 mm platesat 1:50, 1:100 and 1:200. The plates were refed at day 5post-transfection with fresh selection media.

[0196] Screening Colonies

[0197] Approximately 10-12 days post-transfection, one 150 mm culturedish of methotrexate resistant colonies was chosen from eachtransfection, the media aspirated, the plates washed with 10 mlserum-free ESTEP 2 media (668.7 g/50L DMEM (Gibco), 5.5 g/50L pyruvicacid, sodium salt 96% (Mallinckrodt), 185.0 g/50L NaHCO₃ (Mallinkrodt),5.0 mg/ml, 25 ml/50L insulin, 10.0 mg/ml and 25 ml/50 L transferrin).The wash media was aspirated and replaced with 5 ml serum-free ESTEP 2.Sterile Teflon mesh (Spectrum Medical Industries, Los Angeles, Calif.)pre-soaked in serum-free ESTEP 2 was then placed over the cells. Asterile nitrocellulose filter pre-soaked in serum-free ESTEP 2 was thenplaced over the mesh. Orientation marks on the nitrocellulose weretransferred to the culture dish. The plates were then incubated for 5-6hours in a 37° C., 5% CO₂ incubator. Following incubation, the filterwas removed, and the media aspirated and replaced with DMEM/5% FBS, 1×PSN (Gibco BRL) media. The filter was then placed into a sealable bagcontaining 50 ml buffer (25 mM Tris, 25 mM glycine, 5 mMβ-mercaptoethanol) and incubated in a 65° C. water bath for 10 minutes.The filters were blocked in 10% nonfat dry milk/PBS, 0.1% PBS (Sigma)for 15 minutes at room temperature on a rotating shaker. The filter wasthen incubated with an anti-Glu-Glu antibody-HRP conjugate at a 1:1000dilution in 10% nonfat dry milk, 0.1% PBS, 0.1% TWEEN, overnight at 4°C. on a rotating shaker. The filter was then washed three times at roomtemperature in PBS plus 0.1% Tween 20, 5-15 minutes per wash. The filterwas developed with ECL reagent (Amersham Corp., Arlington Heights, Ill.)according the manufacturer's directions and exposed to film (HyperfilmECL, Amersham) for approximately 35 seconds.

[0198] The film was aligned with the plate containing the colonies.Using the film as a guide, suitable colonies were selected. Sterile, 3mm coloning discs (PGC Scientific Corp., Frederick, Md.) were soaked intrypsin, and placed on the colonies. Twelve colonies for each constructwere transferred into 200 μl of selection medium in a 96 well plate. Aseries of seven, two-fold dilutions were carried out for each colony.The cells were grown for one week at 37° C. at which time the wellswhich received the lowest dilution of cells which are now at the optimumdensity were selected, trypsinized and transferred to a 12 well platecontaining selection media. The 150 mm culture dish was also trypsinizedand the remainder of the cells were pooled and subjected to Western Blotanalysis and mycoplasma testing. The pool was frozen for storage.

[0199] The clones were expanded directly from the 12 well plate into twoT-75 flasks each. One flask was kept to continue cell growth, the secondflask was grown in serum-free ESTEP 2 which was harvested for WesternBlot analysis. Clones of each of the expression constructs, based onWestern blot analysis, were selected, pooled and transferred to largescale culture.

EXAMPLE 6 Large Scale Mammalian Expression of zsig39CEE and zsig39NEE

[0200] One T-162 flask, containing confluent cells expressing zsig39CEEand one containing zsig39NEE obtained from the expression proceduredescribed above, were expanded into six T-162 flasks each. One of thesix resulting flasks was used to freeze down four cryovials, and theother five flasks were used to generate a Nunc cell factory.

[0201] The cells from the five T-162 flasks of zsig39CEE and zsig39NEEwere used to independently seed two Nunc cell factories (10 layers,commercially available from VWR). Briefly, the cells from the T-162flasks described above were detached using trypsin, pooled, and added to1.5 liters ESTEPI media (668.7 g/50L DMEM (Gibco), 5.5 g/50L pyruvicacid, sodium salt 96% (Mallinckrodt), 185.0 g/50L NaHCO₃ (Mallinkrodt),5.0 mg/ml and 25 ml/50L insulin (JRH Biosciences), 10.0 mg/ml and 25ml/50L transferrin (JRH Biosciences), 2.5L/50L fetal bovine serum(characterized) (Hyclone), 1 μM MTX, with pH adjusted to 7.05 +/−0.05)prewarmed to 37° C. The media containing the cells was then poured intothe Nunc cell factories via a funnel. The cell factories were placed ina 37° C./5.0% CO₂ incubator.

[0202] At 80-100% confluence, a visual contamination test (phenol redcolor change) was performed on the contents of the Nunc cell factories.Since no contamination was observed, supernatant from the confluentfactories was poured into a small harvest container, sampled anddiscarded. The adherent cells were then washed once with 400 ml PBS. Todetach the cells from the factories, 100 mls of trypsin was added toeach and removed and the cells were then incubated for 2 or 5 minutes inthe residual trypsin. The cells were collected in two, 200 ml washeswith ESTEP1 media. To each of ten ESTEP1 media-containing bottles (1.5liters each, at 37° C.) was added 40 mls of collected cells. One 1.5liter bottle was then used to fill one Nunc factory. Each cell factorywas placed in a 37° C./5.0% CO₂ incubator.

[0203] At 80-90% confluence, a visual contamination test (phenol redcolor change) was performed on the Nunc cell factories. Since nocontamination was observed, supernatant from the confluent factories waspoured into a small harvest container, sampled and discarded. Cells werethen washed once with 400 ml PBS. 1.5 liters of ESTEP2 media (668.7g/50L DMEM (Gibco), 5.5 g/50L pyruvic acid, sodium salt 96%(Mallinckrodt), 185.0 g/50L NaHCO₃ (Mallinkrodt), 5.0 mg/ml, 25 ml/50Linsulin, 10.0 mg/ml and 25 ml/50L transferrin) was added to each Nunccell factory. The cell factories were incubated at 37° C./5.0% CO₂.

[0204] At approximately 48 hours a visual contamination test (phenol redcolor change) was performed on the Nunc cell factories. Supernatant fromeach factory was poured into small harvest containers. Fresh serum-freemedia (1.5 liters) was poured into each Nunc cell factory, and thefactories were incubated at 37° C./5.0% CO₂. One ml of supernatantharvest for each construct was transferred to a microscope slide, andsubjected to microscopic analysis for contamination. The contents of thesmall harvest containers for each construct were pooled and immediatelyfiltered. A second harvest was then performed, substantially asdescribed above at 48 hours and the cell factories were discardedthereafter. An aseptically assembled filter train apparatus was used foraseptic filtration of the harvest supernatant (conditioned media).Assembly was as follows: tubing was wire-tied to an Opti-Cap filter(Millipore Corp., Bedford, Mass.) and a Gelman Supercap 50 filter(Gelman Sciences, Ann Arbor, Mich.). The Supercap 50 filter was alsoattached to a sterile capped container located in a hood; tubing locatedupstream of the Millipore Opti-cap filter was inserted into aperistaltic pump; and the free end of the tubing was placed in the largeharvest container. The peristaltic pump was run between 200 and 300 rpm,until all of the conditioned media passed through the 0.22 μm finalfilter into a sterile collection container. The filtrate was placed in a4° C. cold room pending purification. The media samples 25 saved fromthe various time points were concentrated 10× with a Millipore 5 kDA cutoff concentrator (Millipore Corp., Bedford, Mass.) according tomanufacturer's direction and subjected to Western Blot analysis.Variation in the mobility of the standards is likely responsible for theapparent size difference between the two preparations.

[0205] Zsig39CEE:

[0206] 5 T-162 Flasks=>0.125 mg/L, 28 kDa;

[0207] 1 Factory, FBS=>0.125 mg/L, 28 kDa;

[0208] 10 Factories, FBS=>0.125 mg/L, 28 kDa;

[0209] 10 Factories (#1), SF=>0.125 mg/L, 28 kDa; and

[0210] 10 Factories (#2), SF=>0.125 mg/L, 28 kDa

[0211] Zsig39NEE:

[0212] 5 T-162 Flasks=0.14 mg/L, 38 kDa;

[0213] 1 Factory, FBS=1.39 mg/L, 38 kDa;

[0214] 10 Factories, FBS=0.14 mg/L, 38 kDa;

[0215] 10 Factories (#1), SF=1.39 mg/L, 38 kDa; and

[0216] 10 Factories (#2), SF=1.39 mg/L, 38 kDa.

EXAMPLE 7 Purification Conditions for zsig39 NEE and CEE

[0217] Unless otherwise noted, all operations were carried out at 4° C.The following procedure was used for purifying zsig39 containingN-terminal or C-terminal Glu-Glu (EE) tags described above. A total of25 liters of conditioned media from baby hamster kidney (BHK) cells wassequentially sterile filtered through a 4 inch, 0.2 mM Millipore(Bedford, Mass.) OptiCap capsule filter and a 0.2 mM Gelman (Ann Arbor,Mich.) Supercap 50. The material was then concentrated to about 1.3liters using a Millipore ProFlux A30 tangential flow concentrator fittedwith a 3000 kDa cutoff Amicon (Bedford, Mass.) S10Y3 membrane. Theconcentrated material was again sterile-filtered with the Gelman filteras described above. A mixture of protease inhibitors was added to theconcentrated conditioned media to final concentrations of 2.5 mMethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St. Louis,Mo.), 0.001 mM leupeptin (Boehringer-Mannheim, Indianapolis, Ind.),0.001 mm pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc(Boehringer-Mannheim). A 25.0 ml sample of anti-EE Sepharose, preparedas described below, was added to the sample for batch adsorption and themixture was gently agitated on a Wheaton (Millville, N.J.) rollerculture apparatus for 18.0 h at 4° C.

[0218] The mixture was then poured into a 5.0×20.0 cm Econo-Column(Bio-Rad, Laboratories, Hercules, Calif.) and the gel was washed with 30column volumes of phosphate buffered saline (PBS). The unretainedflow-through fraction was discarded. Once the absorbance of the effluentat 280 nM was less than 0.05, flow through the column was reduced tozero and the anti-EE Sepharose gel was washed batch-wise with 2.0 columnvolumes of PBS containing 0.4 mg/ml of EE peptide (AnaSpec, San Jose,Calif.). The peptide used has the sequence Glu-Tyr-Met-Pro-Val-Asp (SEQID NO:27). After 1.0 h at 4° C., flow was resumed and the eluted proteinwas collected. This fraction was referred to as the peptide elution. Theanti-EE Sepharose gel was then washed with 2.0 column volumes of 0.1 Mglycine, pH 2.5, and the glycine wash was collected separately. The pHof the glycine-eluted fraction was adjusted to 7.0 by the addition of asmall volume of 10×PBS and stored at 4° C. for future analysis ifneeded.

[0219] The peptide elution was concentrated to 5.0 ml using a 15,000molecular weight cutoff membrane concentrator (Millipore, Bedford,Mass.) according to the manufacturer's instructions. The concentratedpeptide elution was separated from free peptide by chromatography on a1.5×50 cm Sephadex G-50 (Pharmacia, Piscataway, N.J.) columnequilibrated in PBS at a flow rate of 1.0 ml/min using a BioCad SprintHPLC (PerSeptive BioSystems, Framingham, Mass.). Two ml fractions werecollected and the absorbance at 280 nM was monitored. The first peak ofmaterial absorbing at 280 nM and eluting near the void volume of thecolumn was collected. This fraction was pure zsig39 NEE or zsig39 CEE.The pure material was concentrated as described above, analyzed bySDS-PAGE and Western blotting with anti-Glu-Glu antibodies, and sampleswere taken for amino acid analysis and N-terminal sequencing. Theremainder of the sample was aliquoted, and stored at −80° C. accordingto our standard procedures. The protein concentration of the purifiedzsig39 NEE was 0.65 mg/ml. The protein concentration of zsig39 CEE was0.3 mg/ml.

[0220] Electrophoresis of zsig39 NEE on SDS-PAGE gels in the absence ofreducing agents showed two bands, present in about equimolar amounts, onCoomassie Blue-stained gels of apparent molecular weights ^(˜)50,000 and^(˜)29,000. On western blots these bands showed cross-reactivity withanti-EE antibodies. Three other bands of apparent molecular weights^(˜)150,000, ^(˜)80,000, and ^(˜)60,000 were also observed on westernblots under these conditions. In the presence of reducing agent, theonly band observed on Coomassie Blue stained gels migrated with anapparent molecular weight of 30,000. The intensity of this band wasincreased relative to either band observed on non-reducing gels. The30,000 kDa band also showed cross-reactivity with anti-EE antibodies onwestern blots and was the only cross-reactive protein present. Inaddition, the intensity of this band was increased relative to theintensity of the band under non-reducing conditions. Virtually identicalresults were obtained for zsig39 CEE by SDS-PAGE and western blottingwith anti-EE antibodies.

[0221] Preparation of Anti-EE Sepharose

[0222] A 100 ml bed volume of protein G-Sepharose (Pharmacia,Piscataway, N.J.) was washed 3 times with 100 ml of PBS containing 0.02%sodium azide using a 500 ml Nalgene 0.45 micron filter unit. The gel waswashed with 6.0 volumes of 200 mM triethanolamine, pH 8.2 (TEA, SigmaCo.) and an equal volume of EE antibody solution containing 900 mg ofantibody was added. After an overnight incubation at 4° C., unboundantibody was removed by washing the resin with 5 volumes of 200 mM TEAas described above. The resin was resuspended in 2 volumes of TEA,transferred to a suitable container, and dimethylpimilimidate-2HCl(Pierce), dissolved in TEA, was added to a final concentration of 36mg/ml of gel. The gel was rocked at room temperature for 45 min and theliquid was removed using the filter unit as described above. Nonspecificsites on the gel were then blocked by incubating for 10 min at roomtemperature with 5 volumes of 20 mM ethanolamine in 200 mM TEA. The gelwas washed with 5 volumes of PBS containing 0.02% sodium azide andstored in this solution at 4° C.

EXAMPLE 8 Construction of zsig39 Amino Terminal Glu-Glu Tagged andCarboxy Terminal Glu-Glu Tagged Yeast Expression Vectors

[0223] Expression of zsig39 in Pichia methanolica utilizes theexpression system described in co-assigned WIPO publication WO 97/17450.An expression plasmid containing all or part of a polynucleotideencoding zsig39 is constructed via homologous recombination. Anexpression vector was built from pCZR204 to express C-terminalGlu-Glu-tagged (CEE) zsig39 polypeptides. The pCZR204 vector containsthe AUG1 promoter, followed by the αFpp leader sequence, followed by ablunt-ended Sma I restriction site, a carboxy-terminal peptide tag(Glu-Glu), a translational STOP codon, followed by the AUG1 terminator,the ADE2 selectable marker, and finally the AUG1 3′ untranslated region.Also included in this vector are the URA3 and CEN-ARS sequences requiredfor selection and replication in S. cerevisiae, and the AmpR and colE1ori sequences required for selection and replication in E. coli. Asecond expression vector was built from zCZR204 to express a N-terminalGlu-Glu-tagged (NEE) zsig39 polypeptides. The zCZR204 expression vectoris as described above, having an amino terminal Glu-Glu tag. The zsig39sequence inserted into these vectors begins at residue 19 (Leu) of thezsig39 amino acid sequence (SEQ ID NO:2).

[0224] For each construct two linkers are prepared, and along withzsig39, were homologously recombined into the yeast expression vectorsdescribed above. The untagged N-terminal linker (SEQ ID NO:28) spans 70base pairs of the alpha factor prepro (aFpp) coding sequence on one endand joins it to the 70 base pairs of the amino-terminus coding sequencefrom the mature zsig39 sequence on the other. The NEE-tagged linker (SEQID NO:29) joins Glu-Glu tag (SEQ ID NO:20) between the aFpp codingsequence and the zsig39 sequence. The-untagged C-terminal linker (SEQ IDNO:30) spans about 70 base pairs of carboxy terminus coding sequence ofthe zsig39 on one end with 70 base pairs of AUG1 terminator sequence.The CEE-tagged linker (SEQ ID NO:31) inserts the Glu-Glu tag (SEQ IDNO:20) between the C-terminal end of zsig39 and the AUG1 terminatorregion.

[0225] Construction of the NEE-Tagged-Zsig39 Plasmid

[0226] An NEE-tagged-zsig39 plasmid was made by homologously recombining100 ng of the SmaI digested pCZR204 acceptor vector, 1 μg of Eco RI-BamHI zsig39 cDNA donor fragment, 1 tg NEE-tagged-zsig39 linker (SEQ IDNO:29) and 1 μg of C-terminal untagged linker (SEQ ID NO:30) in S.cerevisiae.

[0227] The NEE-zsig39 linker was synthesized by a PCR reaction. To afinal reaction volume of 100 μl was added 1 pmol each of linkers,ZC13731 (SEQ ID NO:32) and ZC15268 (SEQ ID NO:33), and 100 pmol of eachprimer ZC13497 (SEQ ID NO:34) and ZC15274 (SEQ ID NO:35) , 10 μl of 10×PCR buffer (Boehringer Mannheim), 1 μl Pwo Polymerase (BoehringerMannheim), 10 μl of 0.25 mM nucleotide triphosphate mix (Perkin Elmer)and dH₂O. The PCR reaction was run 10 cycles at 30 seconds at 94° C., 1minute at 50° C. and 1 minute at 72° C., concluded with a 6 minuteextension at 72°. The resulting 144 bp double stranded, NEE-taggedlinker is disclosed in SEQ ID NO:29.

[0228] The C-terminal untagged zsig39 linker was made via a PCR reactionas described using oligonucleotides ZC15273 (SEQ ID NO:36), ZC15724 (SEQID NO:37), ZC15223 (SEQ ID NO:38) and ZC13734 (SEQ ID NO:39). Theresulting 147 bp double stranded, C-terminal untagged linker isdisclosed in SEQ ID NO:30.

[0229] Construction of the CEE-Zsig39 Plasmid

[0230] A CEE-zsig39 plasmid was made by homologously recombining 100 ngof Sma I digested pCZR204 acceptor vector, the 1 μg of Eco RI-Bam HIzsig39 cDNA donor fragment, 1 μg of N-terminal untagged zsig39 linker(SEQ ID NO:28) and 1 μg of CEE-tagged linker (SEQ ID NO:31) in a S.cerevisiae.

[0231] The N-terminal untagged zsig39 linker was made via a PCR reactionas described above using oligonucleotides ZC14822 (SEQ ID NO:40),ZC14821 (SEQ ID NO:41), ZC15269 (SEQ ID NO:42) and ZC15274 (SEQ IDNO:43). The resulting 144 bp double stranded, N-terminal untagged linkeris disclosed in SEQ ID NO:28.

[0232] The CEE-tagged linker was made via a PCR reaction as describedabove using ZC15273 (SEQ ID NO:44), ZC15267 (SEQ ID NO:45), ZC14819 (SEQID NO:49) and ZC14820 (SEQ ID NO:47). The resulting approximately 144 bpdouble stranded, CEE-tagged linker is disclosed in SEQ ID NO:31.

[0233] One hundred microliters of competent yeast cells (S. cerevisiae)was independently combined with 10 μl of the various DNA mixtures fromabove and transferred to a 0.2 cm electroporation cuvette. The yeast/DNAmixtures were electropulsed at 0.75 kV (5 kV/cm), ∞ ohms, 25 μF. To eachcuvette was added 600 μl of 1.2 M sorbitol and the yeast was plated intwo 300 μl aliquots onto two URA D plates and incubated at 30° C.

[0234] After about 48 hours the Ura⁺ yeast transformants from a singleplate were resuspended in 2.5 ml H₂O and spun briefly to pellet theyeast cells. The cell pellet was resuspended in 1 ml of lysis buffer (2%Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Fivehundred microliters of the lysis mixture was added to an Eppendorf tubecontaining 300 μl acid washed glass beads and 200 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, followed by a 5minute spin in a Eppendorf centrifuge as maximum speed. Three hundredmicroliters of the aqueous phase was transferred to a fresh tube and theDNA precipitated with 600 μl ethanol (EtOH), followed by centrifugationfor 10 minutes at 4° C. The DNA pellet was resuspended in 100 μl H₂O.

[0235] Transformation of electrocompetent E. coli cells (DH10B, GibcoBRL) was done with 1 μl yeast DNA prep and 50 pl of DH10B cells. Thecells were electropulsed at 2.0 kV, 25 RF and 400 ohms. Followingelectroporation, 1 ml SOC (2% Bacto™ Tryptone (Difco, Detroit, Mich.),0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 MMMgSO₄, 20 mM glucose) was plated in 250 μl aliquots on four LB AMPplates (LB broth (Lennox), 1.8% Bacto™ Agar (Difco), 100 mg/LAmpicillin).

[0236] Individual clones harboring the correct expression construct forNEE and CEE tagged zsig39 constructs were identified by sequenceanalysis to verify the presence of the zsig39 insert and to confirm thatthe various DNA sequences had been joined correctly to one another. Theinsert of positive clones were subjected to sequence analysis. Largerscale plasmid DNA was isolated using the Qiagen Maxi kit (Qiagen)according to manufacturer's instruction and the DNA was digested withNot I to liberate the Pichia-zsig39 expression cassette from the vectorbackbone. The Not I-restriction digested DNA fragment was thentransformed into the Pichia methanolica expression host, PMAD16. Thiswas done by mixing 100 μl of prepared competent PMAD16 cells with 10 μgof Not I restriction digested zsig39 and transferred to a 0.2 cmelectroporation cuvette. The yeast/DNA mixture was electropulsed at 0.75kV, 25 μF, infinite ohms. To the cuvette was added 1 ml of 1× YeastNitrogen Base and 500 μl aliquots were plated onto two ADE DS (0.056%-Ade -Trp -Thr powder, 0.67% yeast nitrogen base without amino acids, 2%D-glucose, 0.5% 200× tryptophan, threonine solution, and 18.22%D-sorbitol) plates for selection and incubated at 30° C. Clones werepicked and screened via Western blot for high-level zsig39 expression.The resulting NEE-tagged-zsig39 plasmid containing yeast cells weredesignated PMAD16::pCZR206.14.51 and 14.61 and the CEE-tagged-zsig39plasmid containing yeast cells were designated PMAD16::pCZR209#1 and #2.The clones were then subjected to fermentation.

EXAMPLE 9 Purification of zsig39CEE from Pichia methanolica ConditionedMedium

[0237] Unless otherwise noted, all operations were carried out at 4° C.A mixture of protease inhibitors was added to a 3000 ml sample ofconditioned media from Pichia cultures to final concentrations of 2.5 mMethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co.), 0.001 mMleupeptin (Boehringer-Mannheim), 0.001 mM pepstatin(Boehringer-Mannheim) and 0.4 mM Pefabloc (Boehringer-Mannheim). The pHof the media was adjusted to 7.2 with a concentrated solution of NaOH(Sigma Chemical Co.) following the addition of potassium phosphate(Sigma Chemical Co.) to a final concentration of 0.05M. The sample wascentrifuged at 18,000×g for 30 min at 4° C. in a Beckman JLA-10.5 rotor(Beckman Instruments) in a Beckman Avanti J25I centrifuge (BeckmanInstruments) to remove cell debris. To the supernatant fraction wasadded a 50.0 ml sample of anti-EE Sepharose, prepared as describedabove, and the mixture was gently agitated on a Wheaton (Millville,N.J.) roller culture apparatus for 18.0 h at 4° C. The mixture was thenprocessed as described above for zsig39CEE from BHK cells. The purematerial was concentrated as described above, analyzed by SDS-PAGE andWestern blotting with anti-EE antibodies, and samples were taken foramino acid analysis and N-terminal sequencing. The remainder of thesample was aliquoted, and stored at −80° C. according to our standardprocedures.

[0238] On Coomassie Blue-stained SDS-PAGE gels, the preparationcontained two major bands of apparent molecular weights 23,000 and28,000 and two minor components of 21,000 and 45,000. The mobility ofthese bands was the same in the presence and absence of reducing agents.The only band visible on western blots with anti-EE antibodies in theabsence of reducing agents was a protein of apparent molecular weight150,000 (probably IgG that eluted from the anti EE sepharose column).Western blotting with anti-EE antibodies in the presence of reducingagents, in contrast, showed three bands of apparent molecular weights28,000, 24,000, and 23,000. The concentration of zsig39CEE from Pichiamethanolica was 0.35 mg/ml.

EXAMPLE 10 Zsig39 Antibodies

[0239] A polyclonal antibody was prepared by immunizing two female NewZealand white rabbits with the full length zsig39 polypeptide (SEQ IDNO:2). The polypeptide was derived from purified BHK expressed materialdescribed above. The polypeptide was conjugated to the carrier proteinkeyhole limpet hemocyanin (KLH) with gluteraldehyde. The rabbits wereeach given an initial intraperitoneal (ip) injection of 200 μg ofpeptide in Complete Freund's Adjuvant followed by booster ip injectionsof 100 μg peptide in Incomplete Freund's Adjuvant every three weeks.Seven to ten days after the administration of the third boosterinjection, the animals were bled and the serum was collected. Theanimals were then boosted and bled every three weeks.

[0240] The zsig39 specific antibody was purified from the serum using aProtein A Sepharose. The zsig39 antibody can be characterized by anELISA titer check using the polypeptide of SEQ ID NO:2 as an antibodytarget.

EXAMPLE 11 In vivo Administration of zsig39 Via Adenoviral Delivery

[0241] Twenty four male and 24 female C57B16/J mice, approximately 12weeks old (Jackson Labs, Bar Harbor, Me.) were weighed, body temperaturewas measured and food intake monitored daily for four days prior toinjection (days −4 to −1). On day 0, the mice were divided into threegroups and received 0.1 ml virus (AdV-empty 1.8×10¹¹ virus particles/0.1ml or AdV-zsig39-CEE 5×10¹¹ virus particles/0.1 ml) by intravenous tailvein injection, or no injection at all. Injection should result ininfection of the host's liver and expression of virally delivered geneshould commence within 24 hours and continue for 1 to 4 weeks. Threegroups of mice were tested. Group 1, -untreated, n=8 each male andfemale. Group 2, AdV-Empty (empty virus), n=8 each male and female.Group 3, AdV-zsig39 CEE, n=8 each male and female. Production ofadenovirus containing zsig39CEE was done according to the procedure ofBecker et al., Meth. Cell Biol. 43:161-89, 1994 using commerciallyavailable vectors.

[0242] The animals' body temperatures, weights and the weight of foodingested was monitored during the three week study. No difference wasfound between the groups.

[0243] On day 21 the female mice were euthanized and sacrificed bycervical dislocation, and on day 22 the males were. The animals wereexsanguinated and tissues harvested for necropsy.

[0244] The standard serum chemistry panel was done at the time ofsacrifice. Liver, kidney and metabolic parameters were all within normalranges. Total free fatty acids were assayed on the remaining serum fromeach animal. A statistically significant difference in serum Free FattyAcid levels was seen between both female and male mice (p<0.05% forboth) receiving empty virus and those receiving zsig39 encoding virus byDunn's Multiple Comparisons Test. The zsig39 mice had lower levels.Liver, spleen, kidney, thymus, heart and brain were weighed afterremoval. These tissues and femurs were saved for histology.Histopathological analysis of femoral metaphyseal bone marrow revealed adifference between the treatment groups. The mean % of fat score fromthe metaphyseal bone marrow of female zsig39 mice was significantlygreater (p<0.05% by Dunn's Multiple Comparisons Test) than that of thefemale mice receiving the empty adenovirus. No significant observationswere made on the other tissues examined.

[0245] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

1 50 1 1347 DNA Homo sapiens CDS (198)...(926) 1 gaattcggct cgagagggagcgaaccagga ctggggtgac ggcagggcag ggggcgcctg 60 gccggggaga agcgcgggggctggagcacc accaactgga gggtccggag tagcgagcgc 120 cccgaaggag gccatcggggagccgggagg ggggactgcg agaggacccc ggcgtccggg 180 ctcccggtgc cagcgct atgagg cca ctc ctc gtc ctg ctg ctc ctg ggc 230 Met Arg Pro Leu Leu Val LeuLeu Leu Leu Gly 1 5 10 ctg gcg gcc ggc tcg ccc cca ctg gac gac aac aagatc ccc agc ctc 278 Leu Ala Ala Gly Ser Pro Pro Leu Asp Asp Asn Lys IlePro Ser Leu 15 20 25 tgc ccg ggg cac ccc ggc ctt cca ggc acg ccg ggc caccat ggc agc 326 Cys Pro Gly His Pro Gly Leu Pro Gly Thr Pro Gly His HisGly Ser 30 35 40 cag ggc ttg ccg ggc cgc gat ggc cgc gac ggc cgc gac ggcgcg ccc 374 Gln Gly Leu Pro Gly Arg Asp Gly Arg Asp Gly Arg Asp Gly AlaPro 45 50 55 ggg gct ccg gga gag aaa ggc gag ggc ggg agg ccg gga ctg ccggga 422 Gly Ala Pro Gly Glu Lys Gly Glu Gly Gly Arg Pro Gly Leu Pro Gly60 65 70 75 cct cga ggg gac ccc ggg ccg cga gga gag gcg gga ccc gcg gggccc 470 Pro Arg Gly Asp Pro Gly Pro Arg Gly Glu Ala Gly Pro Ala Gly Pro80 85 90 acc ggg cct gcc ggg gag tgc tcg gtg cct ccg cga tcc gcc ttc agc518 Thr Gly Pro Ala Gly Glu Cys Ser Val Pro Pro Arg Ser Ala Phe Ser 95100 105 gcc aag cgc tcc gag agc cgg gtg cct ccg ccg tct gac gca ccc ttg566 Ala Lys Arg Ser Glu Ser Arg Val Pro Pro Pro Ser Asp Ala Pro Leu 110115 120 ccc ttc gac cgc gtg ctg gtg aac gag cag gga cat tac gac gcc gtc614 Pro Phe Asp Arg Val Leu Val Asn Glu Gln Gly His Tyr Asp Ala Val 125130 135 acc ggc aag ttc acc tgc cag gtg cct ggg gtc tac tac ttc gcc gtc662 Thr Gly Lys Phe Thr Cys Gln Val Pro Gly Val Tyr Tyr Phe Ala Val 140145 150 155 cat gcc acc gtc tac cgg gcc agc ctg cag ttt gat ctg gtg aagaat 710 His Ala Thr Val Tyr Arg Ala Ser Leu Gln Phe Asp Leu Val Lys Asn160 165 170 ggc gaa tcc att gcc tct ttc ttc cag ttt ttc ggg ggg tgg cccaag 758 Gly Glu Ser Ile Ala Ser Phe Phe Gln Phe Phe Gly Gly Trp Pro Lys175 180 185 cca gcc tcg ctc tcg ggg ggg gcc atg gtg agg ctg gag cct gaggac 806 Pro Ala Ser Leu Ser Gly Gly Ala Met Val Arg Leu Glu Pro Glu Asp190 195 200 caa gtg tgg gtg cag gtg ggt gtg ggt gac tac att ggc atc tatgcc 854 Gln Val Trp Val Gln Val Gly Val Gly Asp Tyr Ile Gly Ile Tyr Ala205 210 215 agc atc aag aca gac agc acc ttc tcc gga ttt ctg gtg tac tccgac 902 Ser Ile Lys Thr Asp Ser Thr Phe Ser Gly Phe Leu Val Tyr Ser Asp220 225 230 235 tgg cac agc tcc cca gtc ttt gct tagtgcccac tgcaaagtgagctcatgctc 956 Trp His Ser Ser Pro Val Phe Ala 240 tcactcctag aaggagggtgtgaggctgac aaccaggtca tccaggaggg ctggcccccc 1016 tggaatattg tgaatgactagggaggtggg gtagagcact ctccgtcctg ctgctggcaa 1076 ggaatgggaa cagtggctgtctgcgatcag gtctggcagc atggggcagt ggctggattt 1136 ctgcccaaga ccagaggagtgtgctgtgct ggcaagtgta agtcccccag ttgctctggt 1196 ccaggagccc acggtggggtgctctcttcc tggtcctctg cttctctgga tcctccccac 1256 cccctcctgc tcctggggccggcccttttc tcagagatca ctcaataaac ctaagaaccc 1316 tcaaaaaaaa aaaaaaaaaaagggcggccg c 1347 2 243 PRT Homo sapiens 2 Met Arg Pro Leu Leu Val LeuLeu Leu Leu Gly Leu Ala Ala Gly Ser 1 5 10 15 Pro Pro Leu Asp Asp AsnLys Ile Pro Ser Leu Cys Pro Gly His Pro 20 25 30 Gly Leu Pro Gly Thr ProGly His His Gly Ser Gln Gly Leu Pro Gly 35 40 45 Arg Asp Gly Arg Asp GlyArg Asp Gly Ala Pro Gly Ala Pro Gly Glu 50 55 60 Lys Gly Glu Gly Gly ArgPro Gly Leu Pro Gly Pro Arg Gly Asp Pro 65 70 75 80 Gly Pro Arg Gly GluAla Gly Pro Ala Gly Pro Thr Gly Pro Ala Gly 85 90 95 Glu Cys Ser Val ProPro Arg Ser Ala Phe Ser Ala Lys Arg Ser Glu 100 105 110 Ser Arg Val ProPro Pro Ser Asp Ala Pro Leu Pro Phe Asp Arg Val 115 120 125 Leu Val AsnGlu Gln Gly His Tyr Asp Ala Val Thr Gly Lys Phe Thr 130 135 140 Cys GlnVal Pro Gly Val Tyr Tyr Phe Ala Val His Ala Thr Val Tyr 145 150 155 160Arg Ala Ser Leu Gln Phe Asp Leu Val Lys Asn Gly Glu Ser Ile Ala 165 170175 Ser Phe Phe Gln Phe Phe Gly Gly Trp Pro Lys Pro Ala Ser Leu Ser 180185 190 Gly Gly Ala Met Val Arg Leu Glu Pro Glu Asp Gln Val Trp Val Gln195 200 205 Val Gly Val Gly Asp Tyr Ile Gly Ile Tyr Ala Ser Ile Lys ThrAsp 210 215 220 Ser Thr Phe Ser Gly Phe Leu Val Tyr Ser Asp Trp His SerSer Pro 225 230 235 240 Val Phe Ala 3 244 PRT Homo sapiens 3 Met Leu LeuLeu Gly Ala Val Leu Leu Leu Leu Ala Leu Pro Gly His 1 5 10 15 Asp GlnGlu Thr Thr Thr Gln Gly Pro Gly Val Leu Leu Pro Leu Pro 20 25 30 Lys GlyAla Cys Thr Gly Trp Met Ala Gly Ile Pro Gly His Pro Gly 35 40 45 His AsnGly Ala Pro Gly Arg Asp Gly Arg Asp Gly Thr Pro Gly Glu 50 55 60 Lys GlyGlu Lys Gly Asp Pro Gly Leu Ile Gly Pro Lys Gly Asp Ile 65 70 75 80 GlyGlu Thr Gly Val Pro Gly Ala Glu Gly Pro Arg Gly Phe Pro Gly 85 90 95 IleGln Gly Arg Lys Gly Glu Pro Gly Glu Gly Ala Tyr Val Tyr Arg 100 105 110Ser Ala Phe Ser Val Gly Leu Glu Thr Tyr Val Thr Ile Pro Asn Met 115 120125 Pro Ile Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn His Tyr Asp 130135 140 Gly Ser Thr Gly Lys Phe His Cys Asn Ile Pro Gly Leu Tyr Tyr Phe145 150 155 160 Ala Tyr His Ile Thr Val Tyr Met Lys Asp Val Lys Val SerLeu Phe 165 170 175 Lys Lys Asp Lys Ala Met Leu Phe Thr Tyr Asp Gln TyrGln Glu Asn 180 185 190 Asn Val Asp Gln Ala Ser Gly Ser Val Leu Leu HisLeu Glu Val Gly 195 200 205 Asp Gln Val Trp Leu Gln Val Tyr Gly Glu GlyGlu Arg Asn Gly Leu 210 215 220 Tyr Ala Asp Asn Asp Asn Asp Ser Thr PheThr Gly Phe Leu Leu Tyr 225 230 235 240 His Asp Thr Asn 4 245 PRT Homosapiens 4 Met Glu Gly Pro Arg Gly Trp Leu Val Leu Cys Val Leu Ala IleSer 1 5 10 15 Leu Ala Ser Met Val Thr Glu Asp Leu Cys Arg Ala Pro AspGly Lys 20 25 30 Lys Gly Glu Ala Gly Arg Pro Gly Arg Arg Gly Arg Pro GlyLeu Lys 35 40 45 Gly Glu Gln Gly Glu Pro Gly Ala Pro Gly Ile Arg Thr GlyIle Gln 50 55 60 Gly Leu Lys Gly Asp Gln Gly Glu Pro Gly Pro Ser Gly AsnPro Gly 65 70 75 80 Lys Val Gly Tyr Pro Gly Pro Ser Gly Pro Leu Gly AlaArg Gly Ile 85 90 95 Pro Gly Ile Lys Gly Thr Lys Gly Ser Pro Gly Asn IleLys Asp Gln 100 105 110 Pro Arg Pro Ala Phe Ser Ala Ile Arg Arg Asn ProPro Met Gly Gly 115 120 125 Asn Val Val Ile Phe Asp Thr Val Ile Thr AsnGln Glu Glu Pro Tyr 130 135 140 Gln Asn His Ser Gly Arg Phe Val Cys ThrVal Pro Gly Tyr Tyr Tyr 145 150 155 160 Phe Thr Phe Gln Val Leu Ser GlnTrp Glu Ile Cys Leu Ser Ile Val 165 170 175 Ser Ser Ser Arg Gly Gln ValArg Arg Ser Leu Gly Phe Cys Asp Thr 180 185 190 Thr Asn Lys Gly Leu PheGln Val Val Ser Gly Gly Met Val Leu Gln 195 200 205 Leu Gln Gln Gly AspGln Val Trp Val Glu Lys Asp Pro Lys Lys Gly 210 215 220 His Ile Tyr GlnGly Ser Glu Ala Asp Ser Val Phe Ser Gly Phe Leu 225 230 235 240 Ile PhePro Ser Ala 245 5 215 PRT Tamias sibricus 5 Met Pro Ala Gln Arg Gly GlyAla Leu Ser Met Gly Ala Ala Gly Phe 1 5 10 15 Trp Ile Leu Val Leu SerIle Thr Ser Ala Leu Ala Asp Ser Asn Asn 20 25 30 Gln Gly Asn Ser Glu ProCys Gly Pro Pro Gly Pro Pro Gly Pro Pro 35 40 45 Gly Ile Pro Gly Phe ProGly Ala Pro Gly Ala Leu Gly Pro Pro Gly 50 55 60 Pro Pro Gly Val Pro GlyIle Pro Gly Pro Gln Gly Pro Pro Gly Asp 65 70 75 80 Val Glu Lys Cys SerSer Arg Pro Lys Ser Ala Phe Ala Val Lys Leu 85 90 95 Ser Glu Arg Pro ProGlu Pro Phe Gln Pro Ile Val Phe Lys Glu Ala 100 105 110 Leu Tyr Asn GlnGlu Gly His Phe Asn Met Ala Thr Gly Glu Phe Ser 115 120 125 Cys Val LeuPro Gly Val Tyr Asn Phe Gly Phe Asp Ile Arg Leu Phe 130 135 140 Gln SerSer Val Lys Ile Arg Leu Met Arg Asp Gly Ile Gln Val Arg 145 150 155 160Glu Lys Glu Ala Gln Ala Asn Asp Ser Tyr Lys His Ala Met Gly Ser 165 170175 Val Ile Met Ala Leu Gly Lys Gly Asp Lys Val Trp Leu Glu Ser Lys 180185 190 Leu Lys Gly Thr Glu Ser Glu Lys Gly Ile Thr His Ile Val Phe Phe195 200 205 Gly Tyr Leu Leu Tyr Gly Lys 210 215 6 236 PRT Tamiassibricus 6 Met Tyr Glu Ala Gly Lys Arg Ala Ser Phe Met Gly Gly Ala GlyIle 1 5 10 15 Trp Ile Leu Ala Leu Ser Val Leu Met His Val Val Cys SerMet Tyr 20 25 30 Glu Ala Gly Lys Arg Ala Ser Phe Met Gly Gly Ala Gly IleTrp Ile 35 40 45 Leu Ala Leu Ser Val Leu Met His Val Val Cys Ser Asn ValPro Gly 50 55 60 Pro Gln Gly Pro Pro Gly Met Arg Gly Pro Pro Gly Thr ProGly Lys 65 70 75 80 Pro Gly Pro Pro Gly Trp Asn Gly Phe Pro Gly Leu ProGly Pro Pro 85 90 95 Gly Pro Pro Gly Met Thr Val Asn Cys His Ser Lys GlyThr Ser Ala 100 105 110 Phe Ala Val Lys Ala Asn Glu Leu Pro Pro Ala ProSer Gln Pro Val 115 120 125 Ile Phe Lys Glu Ala Leu His Asp Ala Gln GlyHis Phe Asp Leu Ala 130 135 140 Thr Gly Val Phe Thr Cys Pro Val Pro GlyLeu Tyr Gln Phe Gly Phe 145 150 155 160 His Ile Glu Ala Val Gln Arg AlaVal Lys Val Ser Leu Met Arg Asn 165 170 175 Gly Thr Gln Val Met Glu ArgGlu Ala Glu Ala Gln Asp Gly Tyr Glu 180 185 190 His Ile Ser Gly Thr AlaIle Leu Gln Leu Gly Met Glu Asp Arg Val 195 200 205 Trp Leu Glu Asn LysLeu Ser Gln Thr Asp Leu Glu Arg Gly Thr Val 210 215 220 Gln Ala Val PheSer Gly Phe Leu Ile His Glu Asn 225 230 235 7 222 PRT Rattus norvegicus7 Met Pro Ala Pro Gly Arg Gly Pro Arg Gly Pro Leu Leu Ser Met Pro 1 5 1015 Gly Arg Arg Gly Ala Leu Arg Glu Pro Ala Asp Phe Gly Ser Ser Leu 20 2530 Gly Ala Ala Leu Ala Leu Leu Leu Leu Leu Leu Pro Ala Cys Cys Pro 35 4045 Val Lys Met Tyr Glu Ala Gly Lys Arg Ala Ser Phe Met Gly Gly Ala 50 5560 Gly Ile Trp Ile Leu Ala Leu Ser Val Leu Met His Val Val Cys Ser 65 7075 80 Gly Ile Ser Val Arg Ser Gly Ser Ala Lys Val Ala Phe Ser Ala Thr 8590 95 Arg Ser Thr Asn His Glu Pro Ser Glu Met Ser Asn Arg Thr Met Thr100 105 110 Ile Tyr Phe Asp Gln Val Leu Val Asn Ile Gly Asn His Phe AspLeu 115 120 125 Ala Ser Ser Ile Phe Val Ala Pro Arg Lys Gly Ile Tyr SerPhe Ser 130 135 140 Phe His Val Val Lys Val Tyr Asn Arg Gln Thr Ile GlnVal Ser Leu 145 150 155 160 Met Gln Asn Gly Tyr Pro Val Ile Ser Ala PheAla Gly Asp Gln Asp 165 170 175 Val Thr Arg Glu Ala Ala Ser Asn Gly ValLeu Leu Leu Met Glu Arg 180 185 190 Glu Asp Lys Val His Leu Lys Leu GluArg Gly Asn Leu Met Gly Gly 195 200 205 Trp Lys Tyr Ser Thr Phe Ser GlyPhe Leu Val Phe Pro Leu 210 215 220 8 247 PRT Homo sapiens 8 Met Leu LeuLeu Gln Ala Leu Leu Phe Leu Leu Ile Leu Pro Ser His 1 5 10 15 Ala GluAsp Asp Val Thr Thr Thr Glu Glu Leu Ala Pro Ala Leu Val 20 25 30 Pro ProPro Lys Gly Thr Cys Ala Gly Trp Met Ala Gly Ile Pro Gly 35 40 45 His ProGly His Asn Gly Thr Pro Gly Arg Asp Gly Arg Asp Gly Thr 50 55 60 Pro GlyGlu Lys Gly Glu Lys Gly Asp Ala Gly Leu Leu Gly Pro Lys 65 70 75 80 GlyGlu Thr Gly Asp Val Gly Met Thr Gly Ala Glu Gly Pro Arg Gly 85 90 95 PhePro Gln Thr Pro Gly Arg Lys Gly Glu Pro Gly Glu Ala Ala Tyr 100 105 110Met Tyr Arg Ser Ala Phe Ser Val Gly Leu Glu Thr Arg Val Thr Val 115 120125 Pro Asn Val Pro Ile Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn 130135 140 His Tyr Asp Gly Ser Thr Gly Lys Phe Tyr Cys Asn Ile Pro Gly Leu145 150 155 160 Tyr Tyr Phe Ser Tyr His Ile Thr Val Tyr Met Lys Asp ValLys Val 165 170 175 Ser Leu Phe Lys Lys Asp Lys Ala Val Leu Phe Thr TyrAsp Gln Tyr 180 185 190 Gln Glu Lys Asn Val Asp Gln Ala Ser Gly Ser ValLeu Leu His Leu 195 200 205 Glu Val Gly Asp Gln Val Trp Leu Gln Val TyrGly Asp Gly Asp His 210 215 220 Asn Gly Leu Tyr Ala Asp Asn Val Asn AspSer Thr Phe Thr Gly Phe 225 230 235 240 Leu Leu Tyr His Asp Thr Asn 2459 4517 DNA Homo sapiens 9 ctgattccat accagagggg ctcaggatgc tgttgctgggagctgttcta ctgctattag 60 ctctgcccgg gcatgaccag gaaaccacga ctcaagggcccggagtcctg cttcccctgc 120 ccaagggggc ctgcacaggt tggatggcgg gcatcccagggcatccgggc cataatgggg 180 ccccaggccg tgatggcaga gatggcaccc ctggtgagaagggtgagaaa ggagatccag 240 gtcttattgg tcctaaggga gacatcggtg aaaccggagtacccggggct gaaggtcccc 300 gaggctttcc gggaatccaa ggcaggaaag gagaacctggagaaggtgcc tatgtatacc 360 gctcagcatt cagtgtggga ttggagactt acgttactatccccaacatg cccattcgct 420 ttaccaagat cttctacaat cagcaaaacc actatgatggctccactggt aaattccact 480 gcaacattcc tgggctgtac tactttgcct accacatcacagtctatatg aaggatgtga 540 aggtcagcct cttcaagaag gacaaggcta tgctcttcacctatgatcag taccaggaaa 600 ataatgtgga ccaggcctcc ggctctgtgc tcctgcatctggaggtgggc gaccaagtct 660 ggctccaggt gtatggggaa ggagagcgta atggactctatgctgataat gacaatgact 720 ccaccttcac aggctttctt ctctaccatg acaccaactgatcaccacta actcagagcc 780 tcctccaggc caaacagccc caaagtcaat taaaggctttcagtacggtt aggaagttga 840 ttattattta gttggaggcc tttagatatt attcattcatttactcattc atttattcat 900 tcattcatca agtaacttta aaaaaatcat atgctatgttcccagtcctg gggagcttca 960 caaacatgac cagataactg actagaaaga agtagttgacagtgctattt tgtgcccact 1020 gtctctcctg atgctcatat caatcctata aggcacagggaacaagcatt ctcctgtttt 1080 tacagattgt atcctgaggc tgagagagtt aagtgaatgtctaaggtcac acagtattaa 1140 gtgacagtgc tagaaatcaa acccagagct gtggactttgttcactagac tgtgcccttt 1200 tatagaggta catgttctct ttggagtgtt ggtaggtgtctgtttcccac ctcacctgag 1260 agccattgaa tttgccttcc tcatgaatta aaacctcccccaagcagagc ttcctcagag 1320 aaagtggttc tatgatgaag tcctgtcttg gaaggactactactcaatgg cccctgcact 1380 actctacttc ctcttaccta tgtcccttct catgcctttccctccaacgg ggaaagccaa 1440 ctccatctct aagtgctgaa ctcatccctg ttcctcaaggccacctggcc aggagcttct 1500 ctgatgtgat atccactttt tttttttttt gagatggagtctcactctgt cacccaggct 1560 ggagtacagt gacacgacct cggctcactg cagcctccttctcctgggtc caagcaatta 1620 ttgtgcctca gcctcccgag tagctgagac ttcaggtgcattccaccaca catggctaat 1680 ttttgtattt ttagtagaaa tggggtttcg tcatgttggccaggctggtc tcgaactcct 1740 ggcctaggtg atccacccgc ctcgacctcc caaagtgctgggattacagg catgagccac 1800 catgcccagt cgatatctca ctttttattt tgccatggatgagagtcctg ggtgtgagga 1860 acacctccca ccaggctaga ggcaactgcc caggaaggactgtgcttccg tcacctctaa 1920 atcccttgca gatccttgat aaatgcctca tgaagaccaatctcttgaat cccatatcta 1980 cccagaatta actccattcc agtctctgca tgtaatcagttttatccaca gaaacatttt 2040 cattttagga aatccctggt ttaagtatca atccttgttcagctggacaa tatgaatctt 2100 ttccactgaa gttagggatg actgtgattt tcagaacacgtccagaattt ttcatcaaga 2160 aggtagcttg agcctgaaat gcaaaaccca tggaggaattctgaagccat tgtctccttg 2220 agtaccaaca gggtcaggga agactgggcc tcctgaatttattattgttc tttaagaatt 2280 acaggttgag gtagttgatg gtggtaaaca ttctctcaggagacaataac tccagtgatg 2340 tttttcaaag attttagcaa aaacagagta aatagcattctctatcaata tataaattta 2400 aaaaactatc tttttgctta cagttttaaa ttctgaacaatttctcttat atgtgtattg 2460 ctaatcatta aggtattatt ttttccacat ataaagctttgtctttttgt tgttgttgtt 2520 gtttttaaga tggagtttcc ctctgttgcc aggctagagtgcagtggcat gatctcggct 2580 tactgcaacc tttgcctccc aggtttaagc gattcttctgcctcagcctc ccgagtagct 2640 gggaccacag gtgcctacca ccatgccagg ctaatttttgtatttttagt aaagacaggg 2700 tttcaccata ttggccaggc tggtctcgaa ctcctgaccttgtgatctgc ccgcctccat 2760 tgtgttgtta tttgtgagaa agatagatat gaggtttagagagggatgaa gaggtgagag 2820 taagccttgt gttagtcaga actctgtgtt gtgaatgtcattcacaacag aaaacccaaa 2880 atattatgca aactactgta agcaagaaaa ataaaggaaaaatggaaaca tttattcctt 2940 tgcataatag aaattaccag agttgttctg tctttagataaggtttgaac caaagctcaa 3000 aacaatcaag acccttttct gtatgtcctt ctgttctgccttccgcagtg taggctttac 3060 cctcaggtgc tacacagtat agttctaggg tttccctcccgatatcaaaa agactgtggc 3120 ctgcccagct ctcgtatccc caagccacac catctggctaaatggacatc atgttttctg 3180 gtgatgccca aagaggagag aggaagctct ctttcccagatgccccagca agtgtaacct 3240 tgcatctcat tgctctggct gagttgtgtg cctgtttctgaccaatcact gagtcaggag 3300 gatgaaatat tcatattgac ttaattgcag cttaagttaggggtatgtag aggtattttc 3360 cctaaagcaa aattgggaca ctgttatcag aaataggagagtggatgata gatgcaaaat 3420 aatacctgtc cacaacaaac tcttaatgct gtgtttgagctttcatgagt ttcccagaga 3480 gacatagctg gaaaattcct attgattttc tctaaaatttcaacaagtag ctaaagtctg 3540 gctatgctca cagtctcaca tctggtgggg gtgggctccttacagaacac gctttcacag 3600 ttaccctaaa ctctctgggg cagggttatt cctttgtggaaccagaggca cagagacagt 3660 caactgaggc ccaacagagg cctgagagaa actgaggtcaagatttcagg attaatggtc 3720 ctgtgatgct ttgaagtaca attgtggatt tgtccaattctctttagttc tgtcagcttt 3780 tgcttcatat attttagcgc tctattatta gatatatacatgtttagtat tatgtcttat 3840 tggtgcattt actctcttat cattatgtaa tgtccttctttatctgtgat aattttctgt 3900 gttctgaagt ctactttgtc taaaaataac atacgcactcaacttccttt tctttcttcc 3960 ttcctttctt tcttccttcc tttctttctc tctctctctttccttccttc cttcctcctt 4020 ttctctctct ctctctctct ctctcttttc ttgacagactctcgttctgt ggccctggct 4080 ggagttcagt ggtgtgatct tggctcactg ctacctctaccatgagcaat tctcctgcct 4140 cagcctccca agtagctgga actacaggct catgccactgcgcccagcta atttttgtat 4200 ttttcgtaga gacggggttt caccacattc gtcaggttggtttcaaactc ctgactttgt 4260 gatccacccg cctcggcctc ccaaagtgct gggattacaggcatgagcca tcacacctgg 4320 tcaactttct tttgattagt gtttttgtgg tatatctttttccatcatgt tactttaaat 4380 atatctatat tattgtattt aaaatgtgtt tcttacagactgcatgtagt tgggtataat 4440 ttttatccag tctaaaaata tctgtctttt aattggtgtttagacaattt atatttaata 4500 aaatggtgga atttaaa 4517 10 729 DNA ArtificialSequence Degenerate nucleotide sequence encoding the zsig39 polypeptideof SEQ ID NO2. 10 atgmgnccny tnytngtnyt nytnytnytn ggnytngcng cnggnwsnccnccnytngay 60 gayaayaara thccnwsnyt ntgyccnggn cayccnggny tnccnggnacnccnggncay 120 cayggnwsnc arggnytncc nggnmgngay ggnmgngayg gnmgngayggngcnccnggn 180 gcnccnggng araarggnga rggnggnmgn ccnggnytnc cnggnccnmgnggngayccn 240 ggnccnmgng gngargcngg nccngcnggn ccnacnggnc cngcnggngartgywsngtn 300 ccnccnmgnw sngcnttyws ngcnaarmgn wsngarwsnm gngtnccnccnccnwsngay 360 gcnccnytnc cnttygaymg ngtnytngtn aaygarcarg gncaytaygaygcngtnacn 420 ggnaarttya cntgycargt nccnggngtn taytayttyg cngtncaygcnacngtntay 480 mgngcnwsny tncarttyga yytngtnaar aayggngarw snathgcnwsnttyttycar 540 ttyttyggng gntggccnaa rccngcnwsn ytnwsnggng gngcnatggtnmgnytngar 600 ccngargayc argtntgggt ncargtnggn gtnggngayt ayathggnathtaygcnwsn 660 athaaracng aywsnacntt ywsnggntty ytngtntayw sngaytggcaywsnwsnccn 720 gtnttygcn 729 11 17 DNA Artificial SequenceOligonucleotide ZC447 11 taacaatttc acacagg 17 12 18 DNA ArtificialSequence Oligonucleotide ZC976 12 cgttgtaaaa cgacggcc 18 13 20 DNAArtificial Sequence Oligonucleotide ZC14707 13 cccactggac gacaacaaga 2014 20 DNA Artificial Sequence Oligonucleotide ZC14708 14 agcacactcctctggtcttg 20 15 20 DNA Artificial Sequence Oligonucleotide ZC14760 15ccaatgtagt cacccacacc 20 16 20 DNA Artificial Sequence OligonucleotideZC14758 16 tggtgaacga gcagggacat 20 17 20 DNA Artificial SequenceOligonucleotide ZC14759 17 tccccagtct ttgcttagtg 20 18 18 DNA ArtificialSequence Oligonucleotide ZC15002 18 agggaggtgg ggtagagc 18 19 18 DNAArtificial Sequence Oligonucleotide ZC15003 19 tgggggactt acacttgc 18 206 PRT Artificial Sequence Glu-Glu affinity tag peptide 20 Glu Tyr MetPro Val Asp 1 5 21 26 DNA Artificial Sequence Oligonucleotide ZC15037 21actcattcta gactacagca aagact 26 22 25 DNA Artificial SequenceOligonucleotide ZC15038 22 atgtatggat ccctggacga caaca 25 23 20 DNAArtificial Sequence Oligonucleotide ZC13006 23 ggctgtcctc taagcgtcac 2024 19 DNA Artificial Sequence Oligonucleotide ZC13007 24 aggggtcacagggatgcca 19 25 24 DNA Artificial Sequence Oligonucleotide ZC15609 25ttgtgagaat tcatgaggcc actc 24 26 25 DNA Artificial SequenceOligonucleotide ZC15232 26 attcaaggat ccagcaaaga caggt 25 27 6 PRTArtificial Sequence Glu-Glu peptide 27 Glu Tyr Met Pro Val Asp 1 5 28144 DNA Artificial Sequence N-terminal untagged linker 28 ttattgtttatcaatactac tattgctagc attgctgcta aagaagaagg tgtaagcttg 60 gacaagagagaactggacga caacaagatc cccagcctct gcccggggca ccccggcctt 120 ccaggcacgccgggccacca tggc 144 29 144 DNA Artificial Sequence N-terminal Glu-Glutag linker 29 agcattgctg ctaaagaaga aggtgtaagc ttggacaaga gagaagaagaatacatgcca 60 30 147 DNA Artificial Sequence C-terminal untagged linker30 agcatcaaga cagacagcag gttctccgga tttctggtgt actccgactg gcacagctcc 60ccagtctttg cttagatttc ggctgcctgt ttggatattt ttataatttt tgagagtttg 120ccaactaatg tttttctctt ctatgat 147 31 144 DNA Artificial SequenceC-terminal Glu-Glu tag linker 31 agcatcaaga cagacagcac cttctccggatttctggtgt actccgactg gcacagctcc 60 ccagtctttg ctggagggga ggagtatatgcctatggagt agaattccta gtattctagg 120 gctgcctgtt tggatatttt tata 144 3251 DNA Artificial Sequence Oligonucleotide ZC13731 32 ggtgtaagcttggacaagag agaagaagaa tacatgccaa tggaaggtgg t 51 33 62 DNA ArtificialSequence Oligonucleotide ZC15268 33 tgccccgggc agaggctggg gatcttgttgtcgtccagac caccttccat tggcatgtat 60 tc 62 34 44 DNA Artificial SequenceOligonucleotide ZC13487 34 agcattgctg ctaaagaaga aggtgtaagc ttggacaagagaga 44 35 51 DNA Artificial Sequence Oligonucleotide ZC15274 35catggtggcc cggcgtgcct ggaaggccgg ggtgccccgg gcagaggctg g 51 36 50 DNAArtificial Sequence Oligonucleotide ZC15273 36 catcaagaca gacagcaccttctccggatt tctggtgtac tccgactggc 50 37 65 DNA Artificial SequenceOligonucleotide ZC15724 37 tttctggtgt actccgactg gcacagctcc ccagtctttgcttagaattc ggctgcctgt 60 ttgga 65 38 63 DNA Artificial SequenceOligonucleotide ZC15223 38 tggcaaactc tcaaaaatta taaaaatatc caaacaggcagccctagaat actaggaatt 60 cta 63 39 52 DNA Artificial SequenceOligonucleotide ZC13734 39 atcatagaag agaaaaacat tagttggcaa actctcaaaaattataaaaa ta 52 40 40 DNA Artificial Sequence Oligonucleotide ZC1482240 acggtttatt gtttatcaat actactattg ctagcattgc 40 41 62 DNA ArtificialSequence Oligonucleotide ZC14821 41 tcaatactac tattgctagc attgctgctaaagaagaagg tgtaagcttg gacaagagag 60 aa 62 42 63 DNA Artificial SequenceOligonucleotide ZC15269 42 tgccccgggc agaggctggg gatcttgttg tcgtccagttctctcttgtc caagcttaca 60 cct 63 43 51 DNA Artificial SequenceOligonucleotide ZC15274 43 catggtggcc cggcgtgcct ggaaggccgg ggtgccccgggcagaggctg g 51 44 50 DNA Artificial Sequence Oligonucleotide ZC15273 44catcaagaca gacagcacct tctccggatt tctggtgtac tccgactggc 50 45 68 DNAArtificial Sequence Oligonucleotide ZC15267 45 atttctggtg tactccgactggcacagctc cccagtcttt gctggtggtg aagaatacat 60 gccaatgg 68 46 58 DNAArtificial Sequence Oligonucleotide ZC14819 46 aacaggcagc cctagaatactaggaattct attccattgg catgtattct tcaccacc 58 47 39 DNA ArtificialSequence Oligonucleotide ZC14820 47 attataaaaa tatccaaaca ggcagccctagaatactag 39 48 12 DNA Artificial Sequence Illustrative nucleotidesequence 48 atggcttagc tt 12 49 12 DNA Artificial Sequence Illustrativenucleotide sequence 49 tagcttgagt ct 12 50 12 DNA Artificial SequenceIllustrative nucleotide sequence 50 agccatcagc tg 12

What is claimed is:
 1. An isolated polypeptide comprising a sequence ofamino acid residues that is at least 80% identical to SEQ ID NO:2,wherein said sequence comprises: beta strands corresponding to aminoacid residues 105-109, 128-130, 136-139, 143-146, 164-171, 176-182,187-200, 204-210 and 226-231 of SEQ ID NO:2, wherein the beta strandsare separated by at least two amino acid residues; and a receptorbinding domain comprising amino acid residues 111-135 and 170-174 of SEQID NO:2.
 2. An isolated polypeptide according to claim 2, wherein saidpolypeptide is at least 90% identical to SEQ ID NO:2.
 3. An isolatedpolypeptide according to claim 2, wherein said polypeptide comprises acollagen-like domain having at least 22 collagen repeats.
 4. An isolatedpolypeptide according to claim 2, wherein said polypeptide comprisesresidues 19-243 of SEQ ID NO:2.
 5. An isolated polypeptide according toclaim 1, covalently linked amino terminally or carboxy terminally to amoiety selected from the group consisting of affinity tags, toxins,radionucleotides, enzymes and fluorophores.
 6. An isolated polypeptideselected from the group consisting of: a) a polypeptide having asequence of amino acid residues from amino acid residue 30 to amino acidresidue 95 of SEQ ID NO:2; b) a polypeptide having a sequence of aminoacid residues from amino acid residue 30 to amino acid residue 96 of SEQID NO:2; and c) a polypeptide having a sequence of amino acid residuesfrom amino acid residue 30 to 97 of SEQ ID NO:2; d) a polypeptide havinga sequence of amino acid residues from amino acid residue 30 to aminoacid residue 98 of SEQ ID NO:2; e) a polypeptide having a sequence ofamino acid residues from amino acid residue 98 to amino acid residue 243of SEQ ID NO:2; f) a polypeptide having a sequence of amino acidresidues from amino acid residue 99 to amino acid residue 243 of SEQ IDNO:2; g) a polypeptide having a sequence of amino acid residues fromamino acid residue 30 to amino acid residue 243 of SEQ ID NO:2; and h) apolypeptide having a sequence of amino acid residues that is 90%identical in amino acid sequence to a), b), c), d), e), f), g) or h). 7.A fusion protein consisting essentially of a first portion and a secondportion joined by a peptide bond, said first portion comprising apolypeptide selected from the group consisting of: a) a polypeptidecomprising a sequence of amino acid residues that is at least 80%identical to SEQ ID NO:2, wherein said sequence comprises: beta strandscorresponding to amino acid residues 105-109, 128-130, 136-139, 143-146,164-171, 176-182, 187-200, 204-210 and 226-231 of SEQ ID NO:2, whereinthe beta strands are separated by at least two amino acid residues; anda receptor binding domain comprising amino acid residues 111-135 and170-174 of SEQ ID NO:2; b) a polypeptide comprising a sequence of aminoacid residues as shown in SEQ ID NO:2 from amino acid residue 16 toamino acid residue 243; c) a polypeptide comprising a sequence of aminoacid residues as shown in SEQ ID NO:2 from amino acid residue 1 to aminoacid residue 243; d) a portion of the zsig39 polypeptide as shown in SEQID NO:2 containing the collagen-like domain or a portion of thecollagen-like domain capable of dimerization or oligomerization; e) aportion of the zsig39 polypeptide as shown in SEQ ID NO:2, containingthe globular-like domain or the receptor binding portion of theglobular-like domain; or f) a portion of the zsig39 polypeptide as shownin SEQ ID NO:2, including the collagen-like domain and the globulardomain; and said second portion comprising another polypeptide.
 8. Afusion protein according to claim 7, wherein said first portion isselected from the group consisting of: a) a polypeptide having thesequence of amino acid residue 30 to amino acid residue 95 of SEQ IDNO:2; b) a polypeptide having the sequence of amino acid residue 30 toamino acid residue 96 of SEQ ID NO:2; c) a polypeptide having thesequence of amino acid residue 30 to amino acid residue 97 of SEQ IDNO:2; d) a polypeptide having the sequence of amino acid residue 30 toamino acid residue 98 of SEQ ID NO:2; e) a polypeptide having thesequence of amino acid residue 30 to amino acid residue 243 of SEQ IDNO:2; f) a polypeptide having the sequence of amino acid residue 98 toamino acid residue 243 of SEQ ID NO:2; and g) a polypeptide having thesequence of amino acid residue 99 to amino acid residue 243 of SEQ IDNO:2.
 9. A fusion protein comprising a secretory signal sequence havingthe amino acid sequence of amino acid residues 1-15 or 1-18 of SEQ IDNO:2, wherein said secretory signal sequence is operably linked to anadditional polypeptide.
 10. A pharmaceutical composition comprising apolypeptide according to claim 1, in combination with a pharmaceuticallyacceptable vehicle.
 11. An antibody that specifically binds to anepitope of a polypeptide according to claim
 1. 12. An isolatedpolynucleotide encoding a polypeptide comprising a sequence of aminoacid residues that is at least 80% identical to SEQ ID NO:2, whereinsaid sequence comprises: beta strands corresponding to amino acidresidues 105-109, 128-130, 136-139, 143-146, 164-171, 176-182, 187-200,204-210 and 226-231 of SEQ ID NO:2, wherein the beta strands areseparated by at least two amino acid residues; and a receptor bindingdomain comprising amino acid residues 111-135 and 170-174 of SEQ IDNO:2.
 13. An isolated polynucleotide according to claim 12, wherein saidpolypeptide is at least 90% identical to SEQ ID NO:2.
 14. An isolatedpolynucleotide according to claim 12, wherein said polypeptide comprisesa collagen-like domain having at least 22 collagen repeats.
 15. Anisolated polynucleotide according to claim 12, wherein saidpolynucleotide is DNA.
 16. An isolated polynucleotide selected from thegroup consisting of: a) a sequence of nucleotides from nucleotide 243 tonucleotide 962 of SEQ ID NO:1; b) a sequence of nucleotides fromnucleotide 252 to nucleotide 962 of SEQ ID NO:1; c) a sequence ofnucleotides from nucleotide 285 to nucleotide 482 of SEQ ID NO:1; d) asequence of nucleotides from nucleotide 285 to nucleotide 485 of SEQ IDNO:1; e) a sequence of nucleotides from nucleotide 285 to nucleotide 488of SEQ ID NO:1; f) a sequence of nucleotides from nucleotide 285 tonucleotide 491 of SEQ ID NO:1; g) a sequence of nucleotides fromnucleotide 285 to nucleotide 926 of SEQ ID NO:1; h) a sequence ofnucleotides from nucleotide 491 to nucleotide 926 of SEQ ID NO:1; i) apolynucleotide encoding a polypeptide having a sequence of nucleotidesthat is at least 80% identical in nucleotide sequence to a), b), c), d),e), f), g) and h); j) nucleotide sequences complementary to a), b), c),d), e), f), g), h) or i); and k) degenerate nucleotide sequences of a),b), c) d), e), f), g), h), i) or j).
 17. An isolated polynucleotideencoding a fusion protein consisting essentially of a first portion anda second portion joined by a peptide bond, said first portion isselected from the group consisting of: a) a polypeptide comprising asequence of amino acid residues that is at least 80% identical to SEQ IDNO:2, wherein said sequence comprises: beta strands corresponding toamino acid residues 105-109, 128-130, 136-139, 143-146, 164-171,176-182, 187-200, 204-210 and 226-231 of SEQ ID NO:2, wherein the betastrands are separated by at least two amino acid residues; and areceptor binding domain comprising amino acid residues 111-135 and170-174 of SEQ ID NO:2; b) a polypeptide comprising a sequence of aminoacid residues as shown in SEQ ID NO:2 from amino acid residue 16 toamino acid residue 243; c) a polypeptide comprising a sequence of aminoacid residues as shown in SEQ ID NO:2 from amino acid residue 1 to aminoacid residue 243; d) a portion of the zsig39 polypeptide as shown in SEQID NO:2 containing the collagen-like domain or a portion of thecollagen-like domain capable of dimerization or oligomerization; e) aportion of the zsig39 polypeptide as shown in SEQ ID NO:2, containingthe globular-like domain or an active portion of the globular-likedomain; or f) a portion of the zsig39 polypeptide as shown in SEQ IDNO:2, including the collagen-like domain and the globular domain; andsaid second portion comprising another polypeptide.
 18. An isolatedpolynucleotide encoding a fusion protein comprising a secretory signalsequence having the amino acid sequence of amino acid residues 1-15 or1-18 of SEQ ID NO:2, wherein said secretory signal sequence is operablylinked to an additional polypeptide.
 19. An isolated polynucleotidecomprising the sequence of nucleotide 1 to nucleotide 729 of SEQ IDNO:10.
 20. An expression vector comprising the following operably linkedelements: a transcription promoter; a DNA segment encoding a polypeptideaccording to claim 1; and a transcription terminator.
 21. An expressionvector according to claim 20, wherein said DNA segment encodes apolypeptide that is at least 90% identical to SEQ ID NO:2.
 22. Anexpression vector according to claim 20, wherein said DNA segmentencodes a polypeptide further comprising a collagen-like domain havingat least 22 collagen repeats.
 23. An expression vector according toclaim 20, wherein said DNA segment encodes a polypeptide covalentlylinked amino terminally or carboxy terminally to an affinity tag.
 24. Anexpression vector according to claim 20 wherein said DNA segment furtherencodes a secretory signal sequence operably linked to said polypeptide.25. An expression vector according the claim 20, wherein said secretorysignal sequence comprises residues 1-15 or 1-18 of SEQ ID NO:2.
 26. Acultured cell into which has been introduced an expression vectorcomprising the following operably linked elements: a transcriptionpromoter; a DNA segment encoding a polypeptide according to claim 1; anda transcription terminator; wherein said cell expresses said polypeptideencoded by said DNA segment.
 27. A method of producing a polypeptidecomprising: culturing a cell into which has been introduced anexpression vector comprising the following operably linked elements: atranscription promoter; a DNA segment encoding a polypeptide accordingto claim 1; and a transcription terminator; whereby said cell expressessaid polypeptide encoded by said DNA segment; and recovering saidexpressed polypeptide.
 28. An oligonucleotide probe or primer comprisingat least 14 contiguous nucleotides of a polynucleotide of SEQ ID NO:10or a sequence complementary to SEQ ID NO:10.
 29. A method for modulatingfree fatty acid metabolism by administering a pharmaceutically effectivedose of a polypeptide according to claim
 1. 30. An isolatedpolynucleotide encoding a polypeptide selected from the group consistingof: a) a polypeptide comprising a sequence of amino acid residues fromamino acid residue 30 to amino acid residue 95 of SEQ ID NO:2; b) apolypeptide comprising a sequence of amino acid residues from amino acidresidue 30 to amino acid residue 96 of SEQ ID NO:2; c) a polypeptidecomprising a sequence of amino acid residues from amino acid residue 30to amino acid residue 97 of SEQ ID NO:2; d) a polypeptide comprising asequence of amino acid residues from amino acid residue 98 to amino acidresidue 243 of SEQ ID NO:2; e) a polypeptide comprising a sequence ofamino acid residues from amino acid residue 99 to amino acid residue 243of SEQ ID NO:2; and f) a polypeptide comprising a sequence of amino acidresidues from amino acid residue 30 to amino acid residue 243 of SEQ IDNO:2.
 31. An isolated polynucleotide according to claim 30, wherein saidpolynucleotide is DNA.
 32. An expression vector comprising the followingoperably linked elements: a transcription promoter; a DNA segmentencoding a polypeptide according to claim 30; and a transcriptionterminator.
 33. An expression vector according to claim 32, wherein saidDNA segment encodes a polypeptide covalently linked amino terminally orcarboxy terminally to an affinity tag.
 34. An expression vectoraccording to claim 32 wherein said DNA segment further encodes asecretory signal sequence operably linked to said polypeptide.
 35. Anexpression vector according the claim 32, wherein said secretory signalsequence comprises residues 1-15 or 1-18 of SEQ ID NO:2.
 36. A culturedcell into which has been introduced an expression vector comprising thefollowing operably linked elements: a transcription promoter; a DNAsegment encoding a polypeptide according to claim 30; and atranscription terminator; wherein said cell expresses said polypeptideencoded by said DNA segment.
 37. A method of producing a polypeptidecomprising: culturing a cell into which has been introduced anexpression vector comprising the following operably linked elements: atranscription promoter; a DNA segment encoding a polypeptide accordingto claim 30; and a transcription terminator; whereby said cell expressessaid polypeptide encoded by said DNA segment; and recovering saidexpressed polypeptide.